Human Salty Taste Receptor And Methods Of Modulating Salty Taste Perception

ABSTRACT

Methods for identifying modulators of the epithelial sodium ion channel and for identifying modulators of salty taste perception are described. Also featured are isolated human salty taste receptors, artificial lipid bilayers comprising an epithelial sodium ion channels, and kits for practicing the claimed methods.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is continuation of U.S. application Ser. No.13/008,330, filed Jan. 18, 2011, which is a divisional of U.S.application Ser. No. 11/875,200, filed Oct. 19, 2007, which claimsbenefit to U.S. Provisional Application No. 60/853,290 filed Oct. 19,2006, the entire contents of which are incorporated by reference herein,in their entirety and for all purposes.

FIELD

The invention relates generally to the field of cell biology. Morespecifically, the invention relates to sodium ion channels and theirrole in the recognition of salty taste in humans.

BACKGROUND

Various publications, including patents, published applications,technical articles and scholarly articles are cited throughout thespecification. Each of these cited publications is incorporated byreference herein, in its entirety and for all purposes.

Sodium plays an important role in the body's metabolism, including,among other things, electrical impulse transmission and fluid andelectrolyte homeostasis. In addition, sodium contributes to thedevelopment and stability of flavor in the various foods ingested byanimals, particularly by humans. The sodium ion can inhibit the bittertaste of some stimuli, thereby modifying the taste of food. Thisinhibitory effect of sodium on bitter taste does not depend on thesaltiness of the compound containing the sodium ion, but rather dependson the concentration of the sodium ion.

Excess intake of sodium, however, has been implicated in various diseasestates, including gastric cancer and hypertension. Hypertension is amajor risk factor for heart disease, stroke, and kidney disease. Becauseof the potential negative health effects of excess sodium consumption,the United States FDA recommends that adults limit their intake to lessthan 2400 milligrams of sodium per day. Nevertheless, Americansgenerally far exceed this recommended allowance. As such, variousmedical and scientific groups have recommended drastic reductions insodium intake.

To further the goal of reduced sodium intake, numerous salty tastemimics and salty taste enhancers have been developed. In general, suchmimics have not proven commercially viable as they lack the cleansaltiness of sodium chloride, and most do not affect food flavor assodium salt does.

The dearth of mimics of salty taste, commonly known as salt substitutes,reflects the extreme structural specificity of the taste receptor. Asfar is known, only sodium chloride (NaCl) and lithium chloride (LiCl)impart a true salty taste. Both heavier anions paired with Na and Li,and heavier cations paired with Cl tend to be bitter. The cationspecificity suggests an ion channel, while the chloride effects suggestsparacellular shunts. In addition, the concentration at which NaClimparts a salty taste is above 50 mM, a concentration on the higher endof receptor processes. These two observations—the specificity for Na andLi, and the effective concentration range—are believed to be the key todiscovering the mechanism of salty taste in humans.

Over the past two decades, numerous studies, both qualitative andquantitative, of salt-induced changes in neural activity in the presenceor absence of specific inhibitors and enhancers have led to thesupposition that an epithelial sodium channel (ENaC) acts as the primaryreceptor for saltiness (Brand et al. (1985) Brain Res. 334:207-14;Feigin et al. (1994) Am. J. Physiol. 266(Cell Physiol):C1165-72; and,Brelin et al. (2006) Adv. Otorhinolaryngol. 63:152-90). While the ENaCserves as the salt receptor for many experimental animals (Halpern, B P(1998) Neurosci. Biobehav. Rev. 23(1):5-47), no conclusive evidence hasemerged that the same holds true for human beings. Notably, theinability of amiloride to inhibit sodium-induced salty taste response inhumans suggests that ENaCs are not involved in human salty tasterecognition, at least to the extent observed in other animals.

Because of this discrepancy between human and animal models, thetransduction mechanisms underlying the perception of salty taste inhumans remain under investigation. Sufficient activation of the nerveeventually evokes the sensation of saltiness in the higher corticalareas (Schoenfeld, Mass. et al. (2004) Neuroscience. 127:347-53).

Because of the robust response shown to amiloride by taste cells of manyrodents, the ENaCs in these cells are assumed to be located primarily atthe apical membrane, above the level of the tight junctions. Thislocation makes them susceptible to the action of drugs such asamiloride. It is assumed that amiloride cannot pass the tight junctions.Augmenting the direct mechanism at the apical membrane is a paracellularshunt pathway into the basolateral area of taste buds below the tightjunction level (Mierson, S et al. (1996) J. Neurophysiol. 76:1297-309).Since sodium can pass the tight junctions, the paracellular mechanismshould result in an amiloride insensitive salty taste response. Thehuman salty response may be amiloride-insensitive because the vastmajority of taste cell ENaCs are located below these tight junctions.Other mechanisms for salt perception may exist. These could be entirelydifferent from the ENaC, or an alternative manifestation of the ENaC dueto sodium load or hormonal influences on ENaC expression or composition.

ENaCs comprise a family of cation channel proteins mediating sodiumpermeation in epithelia (Mano, I et al. (1999) Bioessays 21:568-78).Expression cloning originally demonstrated that there are threehomologous genes, each encoding one of the three subunits of thechannel—i.e., alpha (α), beta (β) and gamma (γ) (Canessa, C M et al.(1994) Nature 367:463-7). Co-expression of all three subunits isessential for maximal Na+ channel activity, although the alpha subunitby itself produces a small current. A fourth subunit, delta (δ) waslater cloned and shown to be similar to the alpha subunit bothstructurally and functionally, albeit with a 30-fold lower affinity foramiloride (Waldmann et al. (1995) J. Biol. Chem. 270:27411-4). Thislower amiloride sensitivity is assumed to be reflected in a motif calledthe PreMR2 sequence. The transmembrane topology of the ENaCs comprisestwo hydrophobic transmembrane domains flanking a long extracellularloop, with intracellular amino and carboxyl termini. The subunitstoichiometry of the ENaCs may be species-specific and tissue-specific,since there is evidence for an α2βγ configuration in rats (Firsov et al.(1998) EMBO J. 17:344-52) and an (α)1β(1)γ(1) arrangement in humans(Staruschenko, A (2005) Biophys. J. 88:3966-75).

For improved health and wellness, there is a need to diminish sodiumintake. This need must be balanced with the desire for the taste ofsodium, and the ability of sodium to impart improved flavor in food. Oneattractive means to diminish dietary sodium without sacrificing sodiumflavor is to use modulators of salty taste. Thus, there is a need toestablish the definitive receptor for salty taste perception and for ameans to identify modulators of salty taste perception.

SUMMARY

The invention provides an isolated human salty taste receptor comprisingat least one beta polypeptide subunit, at least one gamma polypeptidesubunit, and at least one delta polypeptide subunit wherein said deltapolypeptide subunit comprises the amino acid sequence of SEQ ID NO:12.In some aspects, the delta polypeptide subunit has the amino acidsequence of SEQ ID NO:9. Also provided is an isolated human salty tastereceptors comprising at least one alpha polypeptide subunit, at leastone beta polypeptide subunit, at least one delta polypeptide subunit,and at least one gamma polypeptide subunit.

The invention also provides a method for identifying modulators ofepithelial sodium ion channels. Such methods include assembling at leastone epithelial sodium ion channel in a lipid membrane (wherein theepithelial sodium ion channel comprises at least three types ofsubunits, which are independently an alpha subunit, a beta subunit, agamma subunit, a delta subunit, and an epsilon subunit); contacting theion channel with a test compound in the presence of sodium ions orlithium ions; and determining a modulation of the biological activity ofthe epithelial sodium ion channel in the presence of the test compoundrelative to the biological activity of the epithelial sodium ion channelin the absence of the test compound. The lipid membrane is preferably anartificial membrane.

In some aspects, the epithelial ion channel comprises one alpha subunit,one beta subunit, and one gamma subunit. In other aspects, theepithelial ion channel comprises one alpha subunit, one beta subunit,one gamma subunit, and one epsilon subunit. In other aspects, theepithelial ion channel comprises two alpha subunits, one beta subunit,and one gamma subunit. In further aspects the epithelial ion channelcomprises three alpha subunits, three beta subunits, and three gammasubunits. Additional aspects include those wherein the epithelial ionchannel comprises one delta subunit, one beta subunit, and one gammasubunit. In other aspects, the epithelial ion channel comprises twodelta subunits, one beta subunit, and one gamma subunit. In stillfurther aspects, the epithelial ion channel comprises two deltasubunits, two beta subunits, and two gamma subunits. In still furtheraspects, the epithelial ion channel comprises three delta subunits,three beta subunits, and three gamma subunits.

In the method for identifying modulators of epithelial sodium channels,the method may further include contacting the epithelial sodium ionchannel with an epithelial sodium ion channel antagonist, such as, butnot limited to chlorhexidine, amiloride, phenamil, benzamil or ahomolog, analog, or derivative thereof.

In the method for identifying modulators of epithelial sodium channels,suitable lipid components for the membrane include at least one ofphosphatidylcholine, phoshpatidylethanolamine, phostphatidylserine,phosphatidylglyine, phosphatidylinositol, sphingomyelin, cholesterol,cardiolipin, or a homolog, analog, or derivative thereof. As such thelipids may be organized as a micelle, liposome, or lipid bilayer.

In some aspects of the method for identifying modulators of epithelialsodium channels, at least two subunits of an epithelial sodium ionchannel are present in the lipid membrane at differing ratios relativeto each other.

In the step for determining a modulation of the biological activity ofthe epithelial sodium ion channel, any suitable means known in the artmay be used, such as, but not limited to, voltage clamping, and/ormeasurement of an indicator dye. The method may be adapted for highthroughput screening.

The method for identifying modulators of epithelial sodium channels thusprovides compounds identified by the method that act as modulators ofthe epithelial sodium channels. These compounds may be formulated intocompositions by admixing the compounds with a pharmaceuticallyacceptable carrier.

In a specific aspect, the invention provides a method for identifyingmodulators of the human salty taste receptor comprising: assembling atleast one salty taste receptor in a lipid membrane, wherein the saltytaste receptor comprises at least one beta subunit, at least one gammasubunit, and at least one delta subunit; contacting the ion channel witha test compound in the presence of sodium ions or lithium ions; anddetermining a modulation of the biological activity of the salty tastereceptor in the presence of the test compound relative to the biologicalactivity of the salty taste receptor in the absence of the testcompound.

In some aspects, the human salty taste receptor comprises one alphasubunit, one beta subunit, and one gamma subunit. In other aspects, thesalty taste receptor comprises one alpha subunit, one beta subunit, onegamma subunit, and one epsilon subunit. In other aspects, the saltytaste receptor comprises two alpha subunits, one beta subunit, and onegamma subunit. In further aspects the salty taste receptor comprisesthree alpha subunits, three beta subunits, and three gamma subunits.Additional aspects include those wherein the salty taste receptorcomprises one delta subunit, one beta subunit, and one gamma subunit. Inother aspects, the salty taste receptor comprises two delta subunits,one beta subunit, and one gamma subunit. In still further aspects, thesalty taste receptor comprises two delta subunits, two beta subunits,and two gamma subunits. In still further aspects, the salty tastereceptor comprises three delta subunits, three beta subunits, and threegamma subunits.

In the method for identifying modulators of the human salty tastereceptor, the delta subunit preferably comprises the amino acid sequenceof SEQ ID NO:12. In some aspects, the delta receptor comprises the aminoacid sequence of SEQ ID NO:9. In the method for identifying modulatorsof the human salty taste receptor, the method may further comprisecontacting the epithelial sodium ion channel with an epithelial sodiumion channel antagonist, such as, but not limited to, chlorhexidine,amiloride, phenamil, benzamil or a homolog, analog, or derivativethereof.

In the method for identifying modulators of the human salty tastereceptor, the lipid membrane may comprise at least one ofphosphatidylcholine, phoshpatidylethanolamine, phostphatidylserine,phosphatidylglyine, phosphatidylinositol, sphingomyelin, cholesterol,cardiolipin, or a homolog, analog, or derivative thereof. The lipids maybe organized as a liposome or lipid bilayer.

In some aspects of the method for identifying modulators of the humansalty taste receptor, at least two subunits of an epithelial sodium ionchannel are present in the lipid membrane at differing ratios relativeto each other. The channels in the membrane preferably comprise at leastone biological activity of a functional human salty taste receptor.

In the step for determining a modulation of the biological activity ofthe salty taste receptor, any suitable means known in the art may beused, such as, but not limited to voltage clamping, and/or measurementof an indicator dye. The method may be adapted for high throughputscreening.

Compounds that modulate human salty taste perception are identified bythe method of the invention and may include, for example, salty tastemimics, enhancers, modifiers, and inhibitors. The invention thusprovides modulators of human salty taste perception which may further beused in compositions by admixing the compounds with a pharmaceuticallyacceptable carrier, or foods and beverages to modulate the salty tasteperception of the food or beverage.

The invention also provides an artificial lipid membrane comprising atleast one type of phospholipid and an epithelial sodium ion channel orspecific ratios of epithelial sodium ion channel subunits wherein thesubunits are selected from the group consisting of alpha subunits, betasubunits, gamma subunits, delta subunits, and epsilon subunits.

The artificial lipid membrane may comprise at least one phospholipidincluding phosphatidylcholine, phoshpatidylethanolamine,phostphatidylserine, phosphatidylglyine, phosphatidylinositol,sphingomyelin, cholesterol, cardiolipin, or a homolog, analog, orderivative thereof. The lipid membrane may be organized, for example, asa liposome or lipid bilayer.

In some aspects, the artificial lipid membrane comprises at least oneepithelial ion channel comprising one alpha subunit, one beta subunit,and one gamma subunit. In other aspects, the epithelial ion channelcomprises one alpha subunit, one beta subunit, one gamma subunit, andone epsilon subunit. In other aspects, the epithelial ion channelcomprises two alpha subunits, one beta subunit, and one gamma subunit.In further aspects the epithelial ion channel comprises three alphasubunits, three beta subunits, and three gamma subunits. Additionalaspects include those wherein the epithelial ion channel comprises onedelta subunit, one beta subunit, and one gamma subunit. In otheraspects, the epithelial ion channel comprises two delta subunits, onebeta subunit, and one gamma subunit. In still further aspects, theepithelial ion channel comprises two delta subunits, two beta subunits,and two gamma subunits. In still further aspects, the epithelial ionchannel comprises three delta subunits, three beta subunits, and threegamma subunits.

The method also provides a method for preparing such artificial lipidmembrane comprising admixing a liposome comprising at least onephospholipid with an epithelial sodium ion channel or specific ratios ofepithelial sodium ion channel subunits wherein the epithelial sodium ionchannel or epithelial sodium ion channel subunits are dissolved in asuitable aqueous buffer comprising at least one detergent, incubatingthe liposome with the epithelial sodium ion channel or epithelial sodiumion channel subunit for a sufficient amount of time, and removing the atleast one detergent.

The method of preparing the artificial lipid membrane may furthercomprise reconstituting the proteo-liposome into a planar lipid bilayer.

The invention further provides a method for identifying modulators ofsalty taste perception comprising: assembling at least one epithelialsodium ion channel in a lipid membrane, wherein the epithelial sodiumion channel comprises at least one of an alpha subunit, a beta subunit,a gamma subunit, a delta subunit, or an epsilon subunit; contacting theion channel with a test compound in the presence of sodium or lithium;determining a modulation of the biological activity of the epithelialsodium ion channel in the presence of the test compound relative to thebiological activity of the epithelial sodium ion channel in the absenceof the test compound; and administering the test compound to a subjectand determining a modulation of salty taste perception in the subjectrelative to the level of salty taste perception in the subject in theabsence of the test compound. Preferably, the epithelial sodium ionchannel comprises at least one beta subunit, at least one gamma subunit,and at least one delta subunit.

In some aspects, the epithelial ion channel comprises one alpha subunit,one beta subunit, and one gamma subunit. In other aspects, theepithelial ion channel comprises one alpha subunit, one beta subunit,one gamma subunit, and one epsilon subunit. In other aspects, theepithelial ion channel comprises two alpha subunits, one beta subunit,and one gamma subunit. In further aspects the epithelial ion channelcomprises three alpha subunits, three beta subunits, and three gammasubunits. Additional aspects include those wherein the epithelial ionchannel comprises one delta subunit, one beta subunit, and one gammasubunit. In other aspects, the epithelial ion channel comprises twodelta subunits, one beta subunit, and one gamma subunit. In stillfurther aspects, the epithelial ion channel comprises two deltasubunits, two beta subunits, and two gamma subunits. In still furtheraspects, the epithelial ion channel comprises three delta subunits,three beta subunits, and three gamma subunits.

In some aspects, the delta subunit comprises the amino acid sequence ofSEQ ID NO:12. In some aspects, the delta subunit comprises the aminoacid sequence of SEQ ID NO:9.

In some aspects, the subject is a human.

The method permits identification of a compound that reacts in vitrowith the human salty taste receptor and which is perceived by subjectsas salty. The invention thus provides such compounds which may be usedin compositions by admixing the compounds with a pharmaceuticallyacceptable carrier, or foods or beverages to modulate the salty tasteperception of the food or beverage. Preferably, the compounds allowperception of salty taste, but which have a reduced effect on bloodpressure as compared to salt and which have no untoward effect on thesubject.

In some aspects, the compounds can be additionally screened by cellbased assays for epithelial sodium channel activity.

The invention also provides kits for identifying modulators of the humansalty taste receptor comprising at least one form of phospholipid;substantially purified epithelial sodium ion channel subunits comprisingalpha subunits, delta subunits, beta subunits, gamma subunits, orepsilon subunits; and optionally comprising an epithelial sodium ionchannel modulator, sodium or lithium, and instructions for using the kitin a method for identifying modulators of the human salty tastereceptor.

The instructions may provide, for example, directions to admix thesubunits in specific ratios to achieve various forms of the epithelialsodium ion channel of interest. In some aspects, at least two subunitsare added to be present at differing ratios relative to each other.

The kit may contain a modulator such as, but not limited to amiloride,phenamil, benzamil, chlorhexidine, or a source of guanidinium ion.

The invention also provides a method of modulating salty tasteperception (either by stimulating salty taste perception or inhibitingsalty taste perception) comprising contacting a human salty tastereceptor with a compound that stimulates salty taste perception whereinthe salty taste receptor comprises at least one beta polypeptidesubunit, at least one gamma polypeptide subunit, and at least one deltapolypeptide subunit wherein said delta polypeptide subunit comprises theamino acid sequence of SEQ ID NO:12, and wherein said compoundspecifically interacts with said delta subunit.

In some aspects, the human salty taste receptor comprises one alphasubunit, one beta subunit, and one gamma subunit. In other aspects, thesalty taste receptor comprises one alpha subunit, one beta subunit, onegamma subunit, and one epsilon subunit. In other aspects, the saltytaste receptor comprises two alpha subunits, one beta subunit, and onegamma subunit. In further aspects the salty taste receptor comprisesthree alpha subunits, three beta subunits, and three gamma subunits.Additional aspects include those wherein the salty taste receptorcomprises one delta subunit, one beta subunit, and one gamma subunit. Inother aspects, the salty taste receptor comprises two delta subunits,one beta subunit, and one gamma subunit. In still further aspects, thesalty taste receptor comprises two delta subunits, two beta subunits,and two gamma subunits. In still further aspects, the salty tastereceptor comprises three delta subunits, three beta subunits, and threegamma subunits.

In some aspects the compound specifically interacts a portion of thedelta subunit containing the amino acid sequence of SEQ ID NO:12. Insome aspects, the compound binds to the portion of the delta subunitcontaining the amino acid sequence of SEQ ID NO:12.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a human taste bud stained by an ATPase histochemicalprocedure.

FIG. 2 shows antibody detection of the second messenger enzyme,phospholipase Cbeta2 (PLCbeta2) using an immunohistochemical procedureon human taste cells. Panel A shows the subset of cells labeled by theantibody. Panel B is a contrast image of the taste bud and of thesurrounding fungiform papillae.

FIGS. 3 (a, b, c, d) shows an alignment of ENaC delta subunit sequencedfrom cDNA of ten individuals (labeled DENACA, DENACD, DENACE, DENACG,DENACH, DENACI, DENACJ, DENACT, DENACTV and DENACW, respectively), ascompared with the GeneBank sequence of the top row (DENACGB). DENACA,DENACD, DENACE, DENACG, DENACH, DENACI, DENACJ, DENACT, DENACTV andDENACW correspond to SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ IDNO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ IDNO:25, and SEQ ID NO:26, respectively.

FIG. 4 shows amiloride inhibition of ENaC of different composition. ENaCcomposed of human delta, beta, gamma was less sensitive to amiloridethan that composed of human alpha, beta, gamma.

FIG. 5 shows immuno-labeling of a subset of cells in a human taste bud.

FIG. 6 shows the capture of an isolated human taste bud cell by amicropipette from an aqueous suspension. The cell thus captured isplaced in an RNA-preserving medium for further study.

FIG. 7 shows an early quantitative RT-PCR of a single cell tracing theamplification of partial transcripts of the ENaC subunits, alpha, beta,gamma, and delta. The result suggests a cell containing equal copies ofdelta, beta, and gamma, with the alpha transcript showing as a genomiccontrol.

FIG. 8 (a, b, c, d) shows an alignment of ENaC gamma subunit sequencedfrom cDNA of ten individuals (labled GENACA, GENACB, GENACD, GENACE,GENACG, GENACH, GENACJ, GENACT, GENACV, and GENACW, respectively)compared with the GeneBank sequence of the top row (GENACGB). GENACA,GENACB, GENACD, GENACE, GENACG, GENACH, GENACJ, GENACT, GENACV, andGENACW correspond to SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ IDNO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ IDNO:35, and SEQ ID NO:36, respectively.

FIG. 9 shows single channel recording of the activity of the catfishputative taste receptor for L-arginine in planar lipid bilayers.Proteoliposomes containing purified receptor protein from catfish tasteepithelium are fused to planar lipid bilayers. Control records (traceshown in part A) were obtained after addition of proteolipisomes to themembrane bathing solution before addition of L-Arg. The addition of 10μM L-Arg to the cis-side of the bilayer evoked regular periodic channelactivity (trace shown in panel B, including the inset that shows thecurrent record at an expanded scale). After several minutes of singlechannel recording, 100 μM D-Arg was added to the cis-side (trace shownin panel C) and activity ceased. Transmembrane potential was −100 mV.Traces shown in all panels are continuous records of that specificcondition.

DETAILED DESCRIPTION

It is to be understood that this invention is not limited to particularmethods, reagents, compounds, compositions, or biological systems, whichcan, of course, vary. It is also to be understood that the terminologyused herein is for the purpose of describing particular aspects only,and is not intended to be limiting.

Various terms relating to the methods and other aspects of the presentinvention are used throughout the specification and claims. Such termsare to be given their ordinary meaning in the art unless otherwiseindicated. Other specifically defined terms are to be construed in amanner consistent with the definition provided herein.

As used in this specification and the appended claims, the singularforms “a,” “an,” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a cell”includes a combination of two or more cells, and the like.

The term “about” as used herein when referring to a measurable valuesuch as an amount, a temporal duration, and the like, is meant toencompass variations of ±20% or ±10%, more preferably ±5%, even morepreferably ±1%, and still more preferably ±0.1% from the specifiedvalue, as such variations are appropriate to perform the disclosedmethods.

As used herein the “Epithelial Sodium Channel” or, as abbreviated,“ENaC,” refers to a multisubunit protein that is responsible for flow ofor transport of sodium ions across specific epithelium or cellmembranes. ENaCs are generally composed of multiple subunits, generallyα, β, γ subunits. There are also δ and ε subunits which may be in someENaCs in specific tissues. The “salty taste receptor” as discoveredherein, is a species of ENaC that is localized in taste cells and in oneaspect is composed of β, γ, and δ subunits.

As used herein, “test compound” refers to any purified molecule,substantially purified molecule, molecules that are one or morecomponents of a mixture of compounds, or a mixture of a compound withany other material that can be analyzed using the methods of the presentinvention. Test compounds can be organic or inorganic chemicals, orbiomolecules, and all fragments, analogs, homologs, conjugates, andderivatives thereof. Biomolecules include proteins, polypeptides,nucleic acids, lipids, monosaccharides, polysaccharides, and allfragments, analogs, homologs, conjugates, and derivatives thereof. Testcompounds can be of natural or synthetic origin, and can be isolated orpurified from their naturally occurring sources, or can be synthesizedde novo. Test compounds can be defined in terms of structure orcomposition, or can be undefined. The compound can be an isolatedproduct of unknown structure, a mixture of several known products, or anundefined composition comprising one or more compounds. Examples ofundefined compositions include cell and tissue extracts, growth mediumin which prokaryotic, eukaryotic, and archaebacterial cells have beencultured, fermentation broths, protein expression libraries, and thelike.

As used herein, the terms “modulate” means any change, increase, ordecrease in the amount, quality, or effect of a particular activity orprotein. “Modulators” refer to any inhibitory or activating moleculesidentified using in vitro and in vivo assays for, e.g., agonists,antagonists, and their homologs, including fragments, variants, andmimetics, as defined herein, that exert substantially the samebiological activity as the molecule. “Inhibitors” or “antagonists” aremodulating compounds that reduce, decrease, block, prevent, delayactivation, inactivate, desensitize, or downregulate the biologicalactivity or expression of a molecule or pathway of interest. “Inducers,”“activators,” or “agonists” are modulating compounds that increase,induce, stimulate, open, activate, facilitate, enhance activation,sensitize, or upregulate a molecule or pathway of interest. In somepreferred aspects of the invention, the level of inhibition orupregulation of the expression or biological activity of a molecule orpathway of interest refers to a decrease (inhibition or downregulation)or increase (upregulation) of greater than from about 50% to about 99%,and more specifically, about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%,58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, ormore. The inhibition or upregulation may be direct, i.e., operate on themolecule or pathway of interest itself, or indirect, i.e., operate on amolecule or pathway that affects the molecule or pathway of interest.

“Pharmaceutically acceptable carrier” refers to a medium that does notinterfere with the effectiveness of the biological activity of theactive ingredient(s) of a composition, and is not toxic to the subjectto which it is administered.

“Ct” or “threshold cycle” refers to the PCR cycle in which a noticeableincrease in reporter fluorescence above a baseline signal is initiallydetected.

“ΔCt” refers to the difference between the Ct of a sample assay and theCt of a control sample. Thus, ΔCt=Ct(target)−Ct(control).

“ΔΔCt” refers to the difference between the average ΔCt value of atarget sample and the average ΔCt for a corresponding calibrator sample.Thus, ΔΔCt(test sample)=AvgΔCt(test sample)−AvgΔCt(calibrator sample).

“Biological activity” as used herein refers to a measurable function ofan ENaC, including but not limited to, maintenance of a sodium gradientacross the membrane, changes in ion flux, changes in membrane potential,current amplitude, voltage gating, sensitivity to chlorhexidine,amiloride, or its analogs, stimulation by bretylium, novobiocin, orguanidinium ions, binding to subunit-specific monoclonal antibodies, andthe like.

The present invention is based on the discovery that the human saltytaste receptor is an epithelial sodium ion channel. It is thus an objectof the present invention to use the precise molar ratios of the ENaCsubunits and to reconstitute the ENaCs in a lipid bilayer in order toidentify compounds that modulate the biological activity of the ENaCs.In particular it is an object of the present invention to use theprecise molar ratios of the salty taste receptor subunits toreconstitute the salty taste receptor in the a lipid bilayer in order toidentify compounds that modulate the biological activity of the saltytaste receptor and to identify compounds that modulate salty tasteperception in human beings. Without intending to be limited to anyparticular theory or mechanism of action, it is believed that a passiveinflux of sodium ions through epithelial sodium channels in certaintaste receptor cells causes a change in intracellular ion balanceleading to a depolarization, ultimately resulting in neurotransmitterrelease, which in turn produces a perception of salty taste.

In one aspect, the invention provides assays to identify compounds thatbind and/or modulate the human salty taste receptor. The methodscomprise assembling at least one epithelial sodium ion channel in alipid membrane, wherein the epithelial sodium ion channel comprises analpha, beta, gamma, or delta subunit, contacting the at least one ionchannel with a test compound in the presence of sodium or lithium, anddetermining a modulation of the biological activity of the at least oneepithelial sodium ion channel in the presence of the test compoundrelative to the biological activity of the at least one subunit in theabsence of the test compound.

Where the biological activity of the sample containing the test compoundis higher than the activity in the sample lacking the test compound, thecompound is an agonist. If the activity of the sample containing thetest compound is lower than the activity in the sample lacking the testcompound, the compound is an antagonist.

Epithelial sodium ion channels are heteromultimeric complexes that arecomprised of different subunits. Various subunits of ENaC have beenidentified, and include, without limitation, the alpha subunit, the betasubunit, the gamma subunit, the delta subunit, and the epsilon subunit.The ENaC subunits may derived from any species, however, mammalian ENaCsubunits are preferred and the most preferred species is human. Examplesof nucleic acid sequences encoding human ENaC subunits and the deducedamino acid sequences are provided herein. Other subunits with amino acidsequences that are substantially homologous or which represent isoformsof the subunit proteins may be used in practicing the invention Aminoacid sequences that are “substantially homologous” are at least proteinsequences that are from about 80% to about 100% identical to thesequence provided herein for the subunit sequence. More preferably, thesequences are about 85% to about 100% identical. Most preferably, thesequences are about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or100% identical to the reference sequence provided herein for thesubunit.

Representative nucleotide sequences encoding human alpha subunit, humanbeta subunit, human gamma subunit, and human delta subunit are providedas SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:7, respectively.The deduced amino acid sequences for human alpha subunit, human betasubunit, human gamma subunit, and human delta subunit are provided asSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, and SEQ ID NO:8, respectively. Ina preferred aspect for the salty taste receptor, the delta subunitcomprises a cysteine at position 532 with respect to SEQ ID NO:8. Thedelta receptor with Cys₅₃₂ is shown in SEQ ID NO:9. Such substitutionmay arise due to a alteration in the triplet codon from tac to tgc (withrespect to that shown in SEQ ID NO:7) and results in a change fromtyrosine (Tyr) to cysteine (Cys).

In some aspects, the ENaC is comprised of at least one alpha subunit, atleast one beta subunit, and at least one gamma subunit (e.g.,(α)1(β)1(γ) 1). In other aspects, the ENaC is comprised of at least onealpha subunit, at least one beta subunit, at least one gamma subunit,and at least one delta subunit. In other aspects, the ENaC is composedof two alpha subunits, one beta subunit, one gamma subunit (α2βγ). Inother aspects, the ENaC is composed of three alpha subunits, three betasubunits, and three gamma subunits (α)3(β)3(γ)3). In another aspect, theENaC comprises an epsilon subunit and at least one other subunit such asan alpha subunit, beta subunit, delta subunit, gamma subunit, orcombinations thereof. In still other aspects the ENaC comprises aplurality of beta subunits. In a preferred aspect, the ENaC is comprisedof at least one beta subunit, at least one gamma subunit, and at leastone delta subunit (the salty taste receptor). The most preferred aspectis an ENaC comprising at least one beta, at least one gamma, and atleast one delta subunit (e.g., (β)1(γ)1)(δ)1 in which the delta subunitcontains Cys₅₃₂.

The various subunits can be present in the ENaC in different ratiosrelative to other subunits. The observed variation may relate to whichtissue the particular ENaC of interest is expressed in. For example, butnot by way of limitation, an ENaC can be comprised of two alpha, onebeta, and one gamma subunit. Thus, in certain aspects of the invention,the ENaC assembled into a lipid membrane is comprised of at least twosubunits that are present in different ratios relative to the othersubunits. In other aspects, the ENaC is comprised of at least twosubunits that are present in the same ratio relative to the othersubunits. The ratios of the ENaC subunits may also vary depending on thetissue in which the ENaCs of interest are expressed. Further, there maybe important sequence variability in the form of each subunit expressedin various tissues. For example, but not by way of limitation, the deltasubunit of ENaC expressed in the salty taste receptor preferably has acysteine in the putative amiloride binding site of delta at position 532of SEQ ID NO:8 (which encodes human delta from kidney). Human kidneydelta has a tyrosine at this position. Thus, when expressing a humansalty taste receptor, it is preferred to use a delta with the putativeamiloride binding site of MGSLCSLWFGA (SEQ ID NO:12) which includesCYS₅₃₂. As this motif is at least a putative site for amiloride binding,other compounds that modulate the human salty taste receptor may alsobind to this site.

In certain aspects of the invention, the lipid membranes produced withthe ENaC subunits in them contain ENaC subunits that form the saltytaste receptor. These salty taste receptors include at least one beta,at least one gamma, and at least one delta subunit. In preferredaspects, the delta subunit comprises Cys₅₃₂. In other aspects, the ENaCcontains subunits selected from alpha, beta, gamma, delta, and epsilon.In some aspects, the ENaC is composed of at least one alpha, at leastone beta, and at least one gamma. In other aspects, the ENaC comprisesat least one epsilon subunit.

The ENaC or the various subunits that are to be assembled into the lipidmembrane can be obtained from any source suitable in the art. Forexample, an ENaC or any subunit thereof can be freshly isolated from anycell that expresses and ENaC, including cell lines and stable celllines. For example, but not by way of limitation, ENaC are expressed inneural tissue, the pancreas, testes, ovaries, tongue, colon, kidneys,lungs, sweat glands, and the like. In some aspects, the ENaC for saltytaste perception is isolated from the papillae of the tongue. In otheraspects, an ENaC or any subunit thereof can be recombinantly expressed,purified and used to reconstitute a lipid membrane to form functionalENaCs.

In certain aspects, each subunit of the ENaC is separately expressed ina recombinant expression system such as, but not limited to bacterialcells, Spodoptera frugiperda cells, mammalian cells, and frog oocytes.The expressed protein is purified by standard biochemical means as iswell-known in the art. Alternatively, expressed protein may beimmunopurified using immobilized antibodies that specifically bind theENaC subunits. Methods for purifying proteins by immunoaffinity (usingantibodies that specifically bind the subunit or ENaC of interest). Inother aspects, the ENaC subunits are expressed as a fusion protein witha polypeptide that allows for rapid purification and subsequent cleavagefrom the expressed protein. Such purification systems include, but arenot limited to the pGEX system (glutathione-S-transferase fusionproteins) and multi-histidine fusion proteins (for nickel bindingaffinity purification). These and other types of purification aredescribed in numerous references and are well-known to those of skill inthe art. In certain preferred aspects, the ENaC subunits are expressedsimultaneously using a baculovirus system and Spodoptera frugiperdacells and membrane fractions are prepared as described in Rao, U.S. etal. (2002) “Activation of Large Conductance Sodium Channels UponExpression of Amiloride-Sensitive Sodium Channel in SF9 Insect Cells” J.Biol. Chem. 277(7):4900-4905.

In certain aspects, the subunits of the ENaC are substantially purifiedprior to incorporation into the membrane. As used herein, “substantiallypurified” refers to subunits that are at least 80% free of contaminatingmaterial (e.g., proteins, polysaccharides, and lipids) derived from thecells from which they are obtained. Preferably, the subunits are atleast about 85% free of contaminating material. More preferably, thesubunits are at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more free of contaminating material.

To recreate a particular ENaC which may be present in a tissue in theartificial membranes of the invention, the ratio of the subunits presentin the ENaC may be determined by quantitative PCR. As the ratio ofprotein subunits of a multimeric receptor often correlates to the amountof mRNA produced in a cell for the given receptor, quantitative PCR canprovide an efficient means of determining the ratio of mRNA present.Protocols for performing quantitative PCR are well known in the art.Further, given the sequences of the ENaCs provided herein and theknowledge in the art and software available for selecting PCRoligonucleotide primers that can specifically and reliably amplifymessages for particular genes, one of skill in the art may easily androutinely perform quantitative PCR on tissue samples and determine theidentity and ratio of the subunits that form a particular ENaC. Assaysfor determining the relative amounts of mRNA are well known in the art.Once the ratio of mRNA is determined, one may extrapolate the amount ofprotein of each subunit that must be added to the membrane to providethe appropriate stoichiometric amounts of protein to form biologicallyactive ENaCs.

The concentration of affinity-purified protein can be determined bymeasuring the total nitrogen content of the protein eluate and comparingthe nitrogen content with the total protein content of the eluate.Nitrogen content can be determined by any means suitable in the art,such as the well-known Kjeldahl Nitrogen Method. Protein concentrationcan be determined, for example, by spectrophotometry whereby a proteinsample is analyzed for its absorption of light at 280 nm to derive anabsorption coefficient. Any means known in the art for assessingconcentration and/or purity of protein may be used.

The invention thus provides artificial membrane systems containingsubstantially purified ENaC protein subunits that assemble intofunctional ENaCs. Specifically, the invention provides artificialmembrane systems containing substantially purified human salty tastereceptor. These membrane systems permit analysis of ENaCs, including,but not limited to the salty taste receptor apart from contaminatingproteins such as endogenous ENaCs. The invention permits the assembly ofENaCs in which the subunits are added at known ratios to permit theassembly of precise ratios of selected subunits. The lipid membrane cancomprise any combination of lipids. Non-limiting examples of suitablelipids include phosphatidylcholine, phoshpatidylethanolamine,phostphatidylserine, phosphatidylglyine, phosphatidylinositol,sphingomyelin, cholesterol, cardiolipin, or a homolog, analog, orderivative thereof. Phospholipids are preferred, and can be obtainedfrom any source suitable in the art. For example, the phospholipids canbe extracted from a cell, or can be synthetic phospholipids, which arecommercially available.

The lipid membrane can be in any conformation or phase, includingwithout limitation, liposomes, a lipid bilayer, or the hexagonal phase.Liposomes and lipid bilayers are particularly preferred.

The effect of the test compound on the biological activity of the ENaCan be determined by any means suitable in the art. The test compound canbe assessed at multiple concentrations. In some aspects, the testcompound is assessed for its ability to modulate at least one biologicalactivity of the ENaC. In preferred aspects, the ENaC is the salty tastereceptor.

The biological activity of the ENaC can be determined by measuring thecurrent of ENaC assembled in the lipid membrane. Voltage clamping is onepreferable technique to measure ENaC current. Voltage clamp techniquesare well known in the art. (Nagel, G et al. (2005) J. Physiol. 564(Pt3):671-82; Staruschenko, A et al. (2004) J. Biol. Chem. 279:27729-34;Tong, Q et al. (2004) J. Biol. Chem. 279:22654-63; Sheng, S et al.(2000) J. Biol. Chem. 275:8572-81). The following parameters can bemeasured using a voltage clamp: single channel conductance, channel opentime, voltage dependence, blockade induced by application of aparticular compound, and activation induced by application of aparticular compound. Other suitable techniques for measuring thebiological activity of ENaCs include flux assays, patch clamping,voltage-sensitive dyes, and ion-sensitive dyes. Preferably, ENaCactivity is measured by membrane electrophysiology or by assessing thechange in fluorescence of a membrane potential dye in response to sodiumor lithium, or analogs thereof (e.g., isotopes). All such assays arewell known in the art. (Gill, S et al. (2003) Assay Drug Dev. Technol.1:709-17, flux assay; Caldwell, R A et al. (2005) Am. J. Physiol. LungCell Mol. Physiol. 288:L813-9, patch clamp). A variety of voltagesensitive dyes are commercially available, including without limitationstyryl dyes, oxonol dyes, and merocyanine-rhodanine dyes. Selection ofthe appropriate voltage sensitive dye is within the relevant skill inthe art. Similarly, a variety of ion sensitive dyes are commerciallyavailable, including single excitation dyes, dual excitation ratiometricdyes, and dual emission ratiometric dyes.

The salty taste receptor is responsive to sodium and lithium ions.However, unlike other ENaCs, the human salty taste receptor is notsensitive to amiloride. Thus, amiloride should not inhibit or stimulatethe salty taste receptor ENaC. Conversely, chlorhexidine acts as aninhibitor of the salty taste response in humans, and may be used inassays to identify salty taste modifiers. One may assess specificity ofstimulation of the salty taste receptor with test compounds by showingthat the effect is inhibited by chlorhexidine. Moreover, test compoundsthat can overcome the effect of chlorhexidine (and stimulate the saltytaste receptors in the membrane systems of the invention) are strongsalty taste enhancers. Basic compounds containing guanidinium ions aswell as certain amines act as salty taste enhancers. These includeguanidine, arginine, and homoarginine. Both L- and D-arginine areequally effective. While not wishing to be bound by any particulartheory of operation, this lack of enantiomeric specificity suggests thatthe primary enhancing effect derives from a compact, basic moiety, inthis case the guanidinium ion. Thus, salty taste receptors in themembrane system of the invention may be stimulated by contacting themwith a source of guanidinium ions. It may be assumed that theseenhancing compounds interact directly with the human salty taste ionchannel, since most sodium channel blockers and enhancers containguanidinium groups that interact with acidic moieties inside the channelpore lumen. Thus, the molecular mechanisms of human salty taste shareselected functional features in common with known sodium channels butalso have unique pharmacological attributes.

Amiloride and amiloride derivatives (e.g., phenamil, benzamil and thelike) may be useful in assessing other ENaCs, such as those containingan alpha subunit. Amiloride and derivatives may also be used in assaysto inhibit the background (endogenous ENaCs) if the purity of thesubunit preparations is low such that host cell ENaCs are contaminatingthe preparation. Thus, in some aspects of the invention, the methodsfurther comprise contacting the ENaC with a sodium ion channelantagonist. Such antagonists are well-known to those of skill in theart. Preferably, the antagonist is amiloride, chlorhexidine, orhomologs, analogs, or derivatives thereof.

The invention also includes within its scope high-throughput screeningassays to identify compounds that modulate the biological activity ofthe salty taste receptor. High-throughput screening assays permitscreening of large numbers of test compounds in an efficient manner. Forexample, but not by way of limitation, lipid membranes comprising anassembled ENaC can be dispersed throughout a multi-well plate such as a96-well microtiter plate. Each well of the microtiter plate can be usedto run a separate assay against a candidate modulator. A microtiterplate permits screening of multiple concentrations of a test compound,multiple test compounds, alone or in combination with other testcompounds, and multiple ENaCs, including ENaCs with varying ratios ofsubdomains as described and exemplified herein under identical assayconditions. In other aspects of the invention, planar lipid bilayerscontaining the ENaC of interest is contacted with a test compound and ameasurement is taken. The solution on one or both sides of the planarbilayer is changed and the bilayer is contacted with a second testcompound. This may be continuously used as a high-throughput assay. Theassays may take place in the presence of additional agonists orantagonists. Data obtained for the test compounds are compared withmeasurements taken in the presence of known agonists or antagonistsand/or to control samples (such as a non-stimulatory/non-inhibitorymedium).

Serial assays may be performed to narrow down the pool of test compoundsthat act as salty taste modifiers. For example, the in vitro assays ofthe invention may be combined with cell-based assays as a secondary orconfirming screening step. Such assays have been described, for example,in published U.S. Patent Application 2005/0059094 to Servant et al.

An additional aspect of the invention features methods for identifyingcompounds that modulate salty taste perception in a subject by acombination of in vitro and in vivo screening assays. In one aspect, atest compound is first screened in vitro to determine its modulatoryeffect on an epithelial sodium ion channel, and then screened further invivo to determine if the compound can modulate, preferably enhance,salty taste perception in a subject.

In one aspect, the in vitro screening assay comprises identifyingmodulators of the human salty taste receptor comprising contacting atest compound with at least one ENaC and determining a decrease in thebiological activity of the ENaC in the presence of the test compoundrelative to the biological activity of the ENaC in the absence of thetest compound. This aspect can be practiced according to the detailsdescribed herein. In one aspect, the in vivo screening assay comprisesidentifying compounds that enhance salty taste perception in a subjectcomprising administering a test compound to the subject and determiningwhether salty taste perception is enhanced in the subject relative tothe level of salty taste perception by the subject in the absence of thetest compound.

For in vivo screening, subjects can be recruited via an InstitutionalReview Board-approved method such as general advertisement in printmedia. Prior to entering the study, each subject provides informedconsent. The participants can be requested to refrain from eating,drinking or chewing gum for at least one hour prior to testing. Subjectscan be paid to participate in the study.

Experimental solutions containing a candidate test compound to beadministered to study subjects can be presented in the form of a binarymixture such as the compound and an inorganic salt such as NaCl.Preliminary experiments can be carried out to establish an appropriateconcentration range for the test compound and inorganic salts. Forexample, four concentrations of the test compound are used with fourconcentrations of the inorganic salts. Aqueous solutions can be preparedto encompass all possible combinations of the concentrations of the testcompound with the inorganic salts.

To assess the salty taste amplifying properties of a given stimulus, anymeans suitable in the art can be used. One non-limiting example of suchmeans is the method of magnitude estimation. Magnitude estimationmeasures ratings of the perceived intensities of saltiness from asample. Subjects participating in saltiness assessments can beinstructed to rate the saltiness or relative saltiness of a solution.Each solution can be sampled by the subject once, twice, three times, ormore.

Prior to sampling a test solution, subjects can be instructed to rinsetheir mouth. For example, subjects can be instructed to rinse with andexpectorate water four times, preferably within a short duration of timesuch as period of approximately two minutes. Test samples and inorganicsalt solutions can then be administered to the subjects, preferably inrandom order, and without replacement. For example, solutions can beprepared in polystyrene medicine cups (Dynarex, N.Y.) in 10 ml aliquots,and administered to the subjects. The subject can be instructed to ratethe relative saltiness of the solution, and the relative saltinessratings for each solution can be arithmetically averaged to yield singleratings of saltiness.

Magnitude estimation may not reveal differences due to variations insubject number use. To eliminate the variance produced by idiosyncraticnumber usage in the magnitude estimation task, the saltiness ratings canbe standardized to the grand arithmetic mean of the saltiness ratings ofNaCl alone in water (averaged across all NaCl concentrations). Eachsubject's mean saltiness rating can be divided into the grand saltinessmean, and the quotient can be used as the multiplicative standardizationfactor for that individual's saltiness rating. This procedure equatesmean saltiness ratings of NaCl in water across subjects.

Analysis of variance (ANOVA) can be conducted on the standardizedrepeated measures data from the magnitude estimation, and post-hocpairwise comparisons can be conducted with Tukey's honest significantdifference (HSD) analysis.

An alternative to magnitude estimation is a forced-ranking procedure,wherein a series of two-alternative forced-choice pairings are used torank the saltiness of aqueous solutions of NaCl in the presence orabsence of a test compound putative enhancer. In this procedure,subjects can be instructed to taste half of the first solution (forexample, 5 ml or 10 ml solution) of the first pair of samples for threeseconds and expectorate. Subjects then rinse twice and taste half of thesecond sample, expectorate, rinse twice and taste the remainder of thetwo solutions using the same procedure. After tasting both solutionstwice, subjects can be asked to indicate which solution they thought wassaltier. If they report that neither solution seemed saltier, subjectscan be asked to guess (forced-choice). The procedure can repeated forall samples.

The saltiness rankings can be calculated based on the number of times aparticular solution is chosen as being saltier than all other solutionsusing the Friedman analysis of pairwise rankings. The Tukey HSD can becalculated to determine if the differences between individual rankingsare significant.

Compounds identified by any of the foregoing inventive screening methodsare contemplated to be within the scope of this invention. Suchcompounds are preferably agonists of ENaCs, more preferably agonists ofthe human salty taste receptor, and even more preferably are enhancersof human salty taste receptors. Such compounds may be formulated as anutraceutical or pharmaceutical composition by admixing such compound inan amount effective to enhance salty taste perception in the subject towhich it is administered and a pharmaceutically or nutraceuticallyacceptable carrier, as described herein.

It is an object of the invention to use the assays to identify compoundsthat are perceived as salty, as well as to identify compounds thatenhance salty taste (such that a reduced amount of sodium or lithium isperceived as a higher concentration of sodium or lithium). The inventionenables the screening of libraries of compounds including natural orsynthetic molecules including, but not limited to proteins, peptides,oligonucleotides, polynucleotides, polysaccharides, lipids, smallorganic molecules, and the like, for their ability to act as saltsubstitutes, salty taste enhancers, or salty taste inhibitors. Theinvention includes salt substitutes, salty taste enhancers, and saltytaste inhibitors identified by the methods of the invention.

Also featured in accordance with the present invention are artificiallipid membranes and methods for preparing the same. The artificial lipidmembranes comprise at least one lipid and an assembled ENaC or at leastone subunit of an ENaC. In preferred aspects, the ENaC is a salty tastereceptor. The lipid membrane can be comprised of any suitable lipid, andare preferably comprised of phospholipids. Suitable lipids include,without limitation, phosphatidylcholine, phoshpatidylethanolamine,phostphatidylserine, phosphatidylglyine, phosphatidylinositol,sphingomyelin, cholesterol, cardiolipin, or a homolog, analog, orderivative thereof, and these can be obtained from any source suitablein the art. The lipid membrane can be in any conformation, andpreferably is a liposome or lipid bilayer.

In one aspect, an artificial lipid membrane is prepared by admixing aliposome that comprises at least one phospholipid with an ENaC or aparticular subunit or subunits of an ENaC that has dissolved in asuitable aqueous buffer. The aqueous buffer comprises at least onedetergent. Suitable detergents are well known in the art, and includewithout limitation, Tween, Triton, CHAPS, sodium cholate, andoctyl-glucoside. After mixing the phospholipids and ENaC or subunitsthereof, the mixture is allowed to incubate for several minutes,preferably at least about 20 minutes, to permit assembly of the ENaCinto a lipid membrane. Following the incubation, the detergent isremoved according to any means suitable in the art, such as thosedescribed and exemplified herein. Other methods known in the art ofpreparing lipids and liposomes containing proteins may be used toproduce the lipids and liposomes containing the ENaC subunits.

In some aspects, the ENaC is assembled into a liposome. The liposome canbe converted into a planar lipid bilayer by use of techniques that arewell known and routine in the art, including those that are describedand exemplified herein. In some aspects, the liposomes contain asubstance other than found in the surrounding milieu. For example, butnot by way of limitation, the liposomes may contain a fluorescentvoltage-sensitive or membrane potential dye that is responsive to sodiumor lithium, to indicate a change in sodium content as a marker of sodiumflow upon stimulation with a test compound.

The invention also features kits for identifying modulators of the humansalty taste receptor. The kits comprise at least one phospholipid, anisolated epithelial sodium ion channel subunit(s), and optionally asource of sodium and/or lithium ions, and instructions for using the kitin a method for identifying modulators of the human salty tastereceptor. In some aspects, the kits optionally comprise an epithelialsodium ion channel antagonist and/or agonist.

The invention provides a method for modulating salty taste perception ina subject by contacting a salty taste receptor with a compound thatspecifically interacts with the putative amiloride-sensitive region ofthe delta subunit that contains Cys₅₃₂. In human subjects, this deltasubunit comprises the amino acid sequence of SEQ ID NO:12. Themodulators may enhance or inhibit salty taste perception by eitherstimulating the receptor or blocking the receptor. The compounds mayinteract with the delta receptor by binding to the receptor, preferablyin the putative amiloride sensitive region having the amino acidsequence of SEQ ID NO:12.

Using computer programs for rational-based drug design that areavailable in the art, molecular modeling may be performed based on theprimary amino acid sequence data available herein and knowledge in theart as to tertiary structure of ion channels to provide a threedimensional model of the human salty taste receptor. Such modelingpermits the rational selection of candidate compounds that will interactwith specific modulatory sites including, as example, the putativeamiloride binding site of the delta subunit, motif SEQ ID NO:12. Thesecompounds, or classes of compounds, will act as salty taste modifiers.Compounds that interact with these regions (e.g., delta subunit SEQ IDNO:12) are useful as modifiers of salty taste perception. Thus, the datapresented herein provide a structural-functional relationship betweenthe subunits comprising the salty taste receptor and the areas of thesubunits that are likely involved in salty taste perception.

The following actual and prophetic examples are provided to describe theinvention in more detail. They are intended to illustrate, not to limitthe invention.

Example 1 Procedure for Obtaining Human Fungiform Papillae and TasteCells

Human fungiform papillae containing taste buds are routinely obtainedfrom the anterior dorsal surface of the tongues of volunteers by a minorsurgical biopsy procedure carried out under local anesthesia. Thegeneral procedure is described in Spielman, A I et al. Collection oftaste tissue from mammals. Experimental Cell Biology of Taste andOlfaction. Spielman A I and Brand J G eds. CRC Press, Boca Raton, Fla.,pp 25-32. Volunteers give informed consent. This procedure has beenreviewed and approved by an Institutional Review Board. The excisedpapillae can be subsequently used either for RNA extraction,immunohistochemistry or in situ hybridization, or in a procedure thatresults in a suspension of isolated taste cells.

RNA extraction, histochemistry an in situ analysis. When used for totalRNA extraction, papillae are immediately subjected to a standardextraction procedure using TRIzol™ reagent (Invitrogen, Carlsbad,Calif.). The RNA extract is treated with DNase to remove most genomicDNA. Any DNA remaining could otherwise yield false positive results insubsequent steps where the use of intron-spanning primers is notpossible. Genomic material, however, is useful in quantitative reversetrancriptase polymerase chain reaction (QRT-PCR) because the single copyof the genomic DNA signals the point of highest sensitivity of the PCR,and provides thereby a convenient end-point for the procedure. Reversetranscription is then performed on the RNA to yield a DNA copy of theRNA, known as complementary DNA or cDNA. This cDNA will used as thesubstrate in the polymerase chain reaction.

Because the fungiform papillae RNA and subsequent cDNA are generally ofhigh quality, the entire coding sequence or open reading frame (ORF) ofthe protein under study can be immediately amplified. Theoligo-nucleotide primers used to effect this amplification are designedbased on the published sequence of the same or similar protein annotatedin GenBank. The PCR reaction products can be analyzed by agarose gelelectrophoresis. This procedure is often used to obtain the entirecoding sequence of a gene known to be expressed in taste bud cells, thefull sequence of which cannot be obtained readily from single cellanalysis.

The excised papillae may also be used for general orimmunohistochemical, or in situ hybridization analysis. Varioustechniques and procedures are available and can be used to fix andprotect the tissue. As an example, FIG. 1 shows a human taste budstained by an ATPase histochemical procedure. FIG. 2 shows antibodydetection of the second messenger enzyme, phospholipase Cbeta2(PLCbeta2) using an immunohistochemical procedure on human taste cells.The procedure is as follows: Histological sections (8 to 10 microns) offungiform papillae were washed three times in 1×PBS for 10 minutes,placed in blocking buffer at room temperature for 4-18 hours. Blockingbuffer was removed and primary antibody (rabbit anti-PLCbeta2) was addedin three concentrations (1:50, 1:100, and 1:200 in buffer). The primaryantibody solution was removed and the slides were washed three times inPBS. The first wash drained immediately while the subsequent washes wereincubated for 10-20 minutes each. Excess fluid was removed and a thesecondary antibody solution (CY3-labeled goat anti-rabbit, 1:1000) wasadded to the sections and the slides were incubated at room temperaturefor 45-120 minutes. The slides were washed three times in PBS. The firstwash drained immediately while the subsequent washes were incubated for10-20 minutes each. The excess fluid was drained, but slides wereallowed to remain wet. Coverslips were placed on the slides and theslides were examined under a fluorescence microscope.

RT PCR for Identifying ENaC Subunits and Sequencing the Same.

Extraction of total RNA from biopsied fungiform papillae is carried outas described above, without DNase treatment, followed by synthesis offirst-strand cDNA. Amplification of ENaC subunits (no more than 500 bpin size) can be performed with the PCR Core System I reagent kit(Promega Corp., Madison Wis.) using primers as above.

If a product of apparently the correct size is obtained, this product isexcised from the gel and purified. The product is then ligated into aplasmid vector to yield a recombinant plasmid which has the gene for thecoding sequence of the protein (e.g., ENaC 6) inserted into it. Therecombinant plasmid is used to transform bacterial cells which, whenprovided with an appropriate growth medium, produce large amounts ofplasmid. Purification of the bacterial culture yields the recombinantplasmid in a pure form, which enables one to get the sequence of theprotein gene from human fungiform papillae. Finally, a bioinformaticanalysis of the sequence, using the BLAST program confirms that thecorrect sequence has indeed been obtained.

Using this procedure, evidence was found for transcripts of four ENaCsubunit types in human fungiform papillae. These subunits are the alpha,beta, gamma, and delta ENaC subunits. The complete ORF of the alphasubunit was rarely observed, but the complete ORF of the other subunitswas nearly always observed. Surprisingly, it was discovered that thedelta subunit of ENaC is present in human fungiform papillae.

Example 2

Identification of the Human Salty Taste Receptor and the Importance ofthe Delta Subunit

In accordance with the present invention, the delta subunit of the ENaCin the fungiform (taste) papillae of humans. The clones in which thesubunit was detected were from pooled cDNA from 3 individuals who agreedto undergo a biopsy procedure to remove several fungiform papillae fromthe anterior dorsal surface of the tongue.

Characteristics of the Delta Subunit.

The delta subunit of the epithelial sodium channel was detected in thefungiform papillae from thirteen individuals by RT-PCR. The detectedfragments were amplified by PCR and subcloned. The polynucleotideencoding delta subunit from these thirteen individuals was then fullysequenced. It was determined that the human delta subunit from fungiformpapillae differed from human delta subunit cloned from kidney in theputative amiloride binding site. The putative amiloride binding sitecontains a tyrosine at amino acid 532 in delta subunit from kidney (SEQID NO:8), but amino acid 532 in delta from fungiform papillae wascysteines in each of the thirteen samples sequenced (SEQ ID NO:9):

TABLE 1  Sequence of putative amiloride Source of delta binding siteKidney delta: MGSLYSLWFGA (SEQ ID NO: 11) Taste delta# 1:MGSLCSLWFGA (SEQ ID NO: 12) Taste delta# 2: MGSLCSLWFGA (SEQ ID NO: 12)Taste delta# 3: MGSLCSLWFGA (SEQ ID NO: 12)

FIG. 3 shows an amino acid sequence alignment of 11 delta subunits,where the first sequence is the GenBank sequence with the other 10sequences being from 10 different individuals. At position 180, apossible polymorphism (R to P) is indicated. Other positions indicatingpossible polymorphisms are with positions 278 (F to I), 355 (S to R),389 (E to Q), and 566 (R to H). Position 566 has also been implicated inamiloride binding. Without intending to be limited to any particulartheory or mechanism of action, the polymorphisms may play a role insensitivity to salty stimuli or may play a role in sensitivity to tastemodulators.

The Y to C change at position 532 is significant as it may help explainwhy rat salty taste receptors are apparently amiloride sensitive whilehuman salty taste receptors are not. As the rat's delta ENaC subunit isa psuedogene, it is not expressed. It is believed that theamiloride-sensitive alpha subunit functions as part of the saltyreceptor in rat. Although this substitution does not significantly alterthe receptor sensitivity and specificity, the pharmacology of thechannel is altered.

While the delta subunit is amiloride sensitive, it is less so than thealpha (FIG. 4). Thus, if the human salty taste receptor ENaC containedthe usual form of delta, it too should show amiloride sensitivity.However, the putative amiloride binding site in delta from human tastecells contains a non-conservative substitution and may therefore have adifferent sensitivity to amiloride than delta subunit in kidney. Withoutintending to be limited to any particular theory or mechanism of action,it delta shows less sensitivity, this observation potentially can beinterpreted to mean that delta is in the human salty taste receptor,particularly because amiloride cannot cross tight junctions. Because ofthe differences between rat and human, the rat is probably not a goodmodel for salty taste perception in humans.

Cellular Specificity of the Human Fungiform Delta ENaC Subunit.

A human taste bud is shown in FIG. 1, wherein an 8 micron section of ahuman fungiform papilla is stained by an ATPase histochemical procedure.The question now became whether some, all, or none of these taste cellsexpressed delta ENaC. To view only those cells expressing the deltasubunit, an antibody to the delta form of human ENaC was raised inrabbits. A representative photograph is shown in FIG. 5. The slide showstissue specific labeling on a subset of cells within a human taste bud.The implication is that the human salty taste receptor is an ENaCcomposed of a multimer of delta, beta, and gamma subunits or of subunitsalpha, delta, beta, and gamma This specificity of delta in the tastecells accompanied by a notable dearth of full-length alpha in these samebuds, suggested that the human salty taste receptor is adelta-containing ENaC and not simply an alpha, beta, gamma ENaC assuggested by others.

Isolation of Human Taste Bud Cells.

A suspension of single isolated taste bud cells was prepared from humanfungiform papillae by incubation of biopsied papillae in acollagenase-based enzymatic procedure, followed by washing of thepapillae to effectively removed enzyme, then trituration of same througha glass pipette. The resulting suspension was enriched for cells of thetaste bud. Individual cells were captured using a glass micropipette(See FIG. 6) and individually placed into a tube containing 2 to 10 μlof RNAlater.

The Delta Subunit is Located in Taste Bud Cells.

cDNA was derived from 7 human fungiform taste cells that wereindividually isolated and captured, as described above, and then pooled.A product of the correct size (˜500 bp) was noted and its identity as ahuman ENaC delta subunit was confirmed by sequencing. Using this PCRprocedure of identifying overlapping segments of the ORF of delta ENaC,the complete ORF of taste cell delta ENaC was obtained.

Single cell RT-PCR using nested primers was also performed, and revealedthat two out of twelve human taste bud cells tested provided strongevidence for expression of delta, beta, and gamma subunits, withoutexpressing full-length alpha (data not shown). One early single cellQ-RT PCR revealed no message for the alpha subunit but approximatelyequal numbers of message copies for delta, gamma and beta (FIG. 7).

Using calcium imaging on a preparation of isolated taste cells, it ispossible to identify those individual cells that are activated by sodiumchloride. These cells are captured and their contents analyzed byQ-SC-RT-PCR. In a group of 30 salt sensitive cells, the primaryexpressed subunit was determined to be delta. Eight expressed delta,beta, and gamma, while 7 expressed delta, alpha, beta, and gamma.

An alignment of the amino acid sequences of the 10 gamma subunitssequenced from taste cells as compared to the GenBank sequence for gammais shown in FIG. 8.

Having identified the salty receptor as delta, beta, gamma or delta,alpha, beta, gamma, each subunit will be expressed and reconstitutedinto lipid bilayers for analyses, as provided by the examples below.

Example 3 Preparation of Liposomes and Artificial Lipid Bilayers

This example demonstrates the techniques that are readily practiced tosolubilize an abundant receptor from its membrane milieu, purify thereceptor, and reconstitute the receptor in an artificial lipid membranesuch as a lipid bilayer. Such membranes serve as an artificialbiological membrane in which the receptor resumes its nativeconformation and can be studied in detail and in isolation, e.g.,without interference from other proteins or the metabolic whimsy of aliving cell.

This example, in part, describes the extraction, purification, andmembrane-reconstitution of a taste receptor for L-arginine (L-arg) fromcatfish. The methods, which are published and described in Grosvenor, Wet al. (2004) BMC Neuroscience 5:25, can be readily adapted forreconstitution of ENaC, ENaC subunits, and salty taste receptors inlipid membranes as described below. The catfish has served as a modelfor taste receptor studies because the receptors are very sensitive tocertain amino acids. One such amino acid is L-arg. Like the ENaC, thetaste receptor for L-arg in catfish is an ion channel. Parallelapproaches are utilized in solubilizing the L-arg and ENaC-typereceptors. The receptors differ primarily by origin—the L-arg receptoris purified from catfish taste tissue and the ENaC subunits aresynthesized by a heterologous cell culture expression system.

Liposome Generation.

Liposomes are used to carry the solubilized receptor to the bilayerconstruct. The major challenge to studying a membrane-soluble protein isdeveloping a procedure to move the protein from its native membrane orsynthesis end point to an artificial lipid bilayer. Solubilizationusually uses detergent and this detergent must be removed to avoiddamage to the bilayer. The liposome performs this transfer by taking upthe protein from the detergent system and giving it up to the bilayer.

Liposomes are prepared by adding 5 mg of the mixture of1,2-dioleoyl-sn-glycero-3-phosphoethanolamine:1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPE:DOPC) in a 2:1 ratio in0.5 ml of chloroform to a round bottom flask. The flask is rotated for30-40 min at 4° C. After evaporation of the chloroform, a thin layer oflipid is formed to which 2 ml of buffer solution (300 mM NaCl, 50 mMTris, pH=7) is added. After addition of the buffer, the flask isbath-sonicated 3 times with 3-5 min pulses to induce liposome formation.Alternatively, the probe sonicator can be pulsed for only 30-40 sec.

Dissolution of the L-ArgR into Liposomes and Pharmacology of L-ArgR in aLipid Membrane.

An amount (0.01 to 0.5 mg) of the L-ArgR dissolved in 100 mM NaCl/50 mMTris, pH=7.1 containing one of several detergents such as Tween, CHAPS,Triton, Sodium Cholate, or/and Octyl-glucoside is added to theliposomes. The ratio of protein to lipid (mass:mass) for measuringchannel activity as single cannel events is 1:2000-5000. The ratio formeasuring macroscopic properties is in the range of 1:50-100. Theprotein-lipid mixture is incubated for 20-30 mins.

The properties of the L-ArgR as measured in the bilayer are very similarto those observed through taste nerve recordings from the animal, whenthe L-ArgR is in its native state. For example, the taste nerverecordings indicate that the sensitivity of the native receptor is inthe tenths of uM of L-arg, while D-arg inhibits the receptor. FIG. 9shows that when the L-ArgR is reconstituted into a bilayer, the sameproperties are seen. This demonstrates that a receptor like the L-ArgRis more directly and more readily studied when in the bilayer than whenin the native state where its activity must be inferred from secondaryneural recordings.

Reconstitution of the ENaC Protein into Liposomes.

An amount (0.01 to 0.5 μg) of the sodium channel of interest, includingENaCα, ENaCδ, or specific ratio mixtures derived from the ENaC subunits,α, β, γ, δ, dissolved in 100 mM NaCl/50 mM Tris, pH=7.1 containing oneof several detergents such as Tween, CHAPS, Triton, Sodium Cholate,or/and Octyl-glucoside will be added to the liposomes. The ratio ofprotein to lipid (mass:mass) for measuring channel activity as singlecannel events is 1:2000-5000. The ratio for measuring macroscopicproperties is in the range of 1:50-100. The protein-lipid mixture willbe incubated for 20-30 mins This procedure will be followed because theENaC protein, being a membrane channel, needs to remain in solutionwhile it is reconstituted into liposomes.

Reconstituted detergent-free proteo-liposomes containing one or more ofthe ENaCs can be prepared at least two ways. In one method, they can beformed through centrifugation of the protein:lipid mixture through gelfiltration columns. These gel columns are prepared from Sephadex G-50(fine), swollen overnight, and poured into 5-ml disposable columns(1.5-ml bed volume). Columns are pre-spun in a centrifuge at ˜1,000×g.The protein:lipid mixture is loaded on the top of the column, andproteo-liposomes free of detergent can be recovered by spinning thecolumns at 700 g for 1 min. Alternatively, detergent can be removed bydialysis. For dialysis, the protein:lipid mixture is loaded into acassette dialysis unit and the mixture dialyzed overnight against 2000ml of a Tris/NaCl/sucrose (detergent-free) buffer at 4° C. Phospholipidvesicles containing the protein are expected to form spontaneously asthe concentration of detergent decreases during the dialysis.

Reconstitution of the Channel Proteins from Proteo-Liposomes into aPlanar Lipid Bilayer.

The planar lipid bilayer is formed on an aperture between two aqueouscompartments, which for operational purposes are called cis and transcompartments. The voltage generator will be connected to the ciscompartment, with an Ag/AgCl electrode, to control membrane potential.The trans compartment (virtual ground) will be connected to the input ofthe current-measuring amplifier through a second Ag/AgCl electrode.

Forming the Bilayer.

A 4:1 mixture of DOPE:DOPC will be dissolved in 25 μl of n-decane(concentration ranging from 15 to 25 mg/ml). This mixture will be keptat room temperature and prepared each day that the experiment isperformed. Electrode compartments will be filled with 3 M KCl and theAg/AgCl electrodes will be placed in the compartment. The cis and transcompartments will be filled with the recording bath solution (100 mMNaCl, 10 Tris, pH 7) and agar bridges will be placed between them andthe electrode compartments. To form the bilayer, a droplet of the lipidmixture will be spread onto the hole from the cis side.

The lipids can be determined to be completely formed around the holewhen the resistance increased and the signal is not saturating. Toverify that an organized bilayer has formed, the voltage pulses acrossthe bilayer can be applied and “capacitive currents” can be measured.For a hole of 100 μm, the capacitance is expected to be of the order of50-100 pF. The electrical resistance of the bilayer is expected to behigher than 109 Ohm.

Reconstitution.

After the bilayer has formed, 10-15 μl of the proteo-liposomes will beadded to the cis side of the bilayer under constant stirring. Whenchannel subunits are incorporated into the bilayer, the currents areexpected to change in steps. Macroscopic current will be measured whenmany channels are incorporated.

Liposome fusion with the bilayer happens spontaneously, and currentswill be able to be recorded within about 5 to 30 minutes. In some cases,it may be necessary to facilitate the liposome fusion by: creating aconcentration gradient across the liposome by adding the liposomesformed previously in 300 mM NaCl to a bilayer bathing solutioncontaining 100 mM NaCl, or by creating a concentration gradient acrossthe lipid bilayer by adding 100 mM NaCl to the cis side and 10 mM NaClto the trans side, or by changing bilayer and/or liposome lipidcomposition by the addition of negatively charged lipid such as DOPS tothe bilayer.

Example 4 Expression of Varying Ratios of ENaC Receptor Subdomains inLipid Bilayers

This is a prophetic example. The ENaC is a heteromultimeric complexgenerally comprised of three subunits: either of subunits α, β, and γ(in most tissues as α2 βγ complex) or subunits δ, β, and γ. Thesesubunits can assemble in varying ratios, often dictated by the tissuesource. Varying the relative ratios of the subunits confers uniquekinetics and pharmacology upon these channels. Without intending to belimited to any particular theory or mechanism of action, it is believedthat the δ subunit replaces the α subunit in many tissues, and that sucha substitution may modify particularly the pharmacology of the channel.

Single Cell Quantitative PCR with Specific Reference to Estimation ofthe Ratios Of ENaC Subunits.

While there is no guarantee of a one-to-one correspondence betweenamount of message and amount of protein, Q-PCR is one tool available forestimation of ENaC ratio. Assuming a salty taste cell is active, it islikely to have a number of copies of a particular subunit. It is likelythat the ratio of message copies will be at least approximately that ofthe protein products. Quantitative single cell PCR can be used to gain asemi-quantitative picture of the relative abundance of message for anyproteins of interest. The procedure, although theoretically straightforward, presents a number of challenging obstacles. With taste cells,for example, RNA quality can be problematic because the time-consumingprocedure currently used to obtain isolated cells (see above) isconducive to destruction of RNA. To be confident in the experimentaltechnique, the following procedure can be carried out: (1) Designseveral unique primer pairs for each gene of interest, using only thosethat have almost identical efficiency under the same PCR conditions forevery gene of interest. (2) Construct a primer set (mixture of primerpairs) from the appropriate pairs above that registers as a blank whenused in a water control PCR reaction. (3) Collect individual cells (asabove) into an RNase-free environment, lyse the cell and reversetranscribe the single cell mRNA content using a commercially availablekit. (4) Run a limited (10-25) number of cycles of the first stage PCRwith the primer set and condition above, so that all of the reactionsare in a linear amplification phase. (5) Dilute the above reaction(100×-1000×), and use an aliquot as template along with a single pair ofprimers from the set above and perform the second stage of PCR (induplicate/triplicate) using a Q-PCR machine. (6) A relativequantification method is used for data analysis. Normalization is basedon amplification of a genomic DNA that is not translated/transcribed ofwhich there is, by definition, one copy of the gene. The differences ingene expression can be determined by comparing ratio (ΔCt between targetgene and genomic reference sequence in sample) differences (ΔΔCts, thedifferences of ΔCts between two samples).

A taste bud cell containing message for human ENaC subunits δ, β, and γ,but no message for α was apparent. The Q-PCR trace of this analysis isshown in FIG. 7. From this single cell, it can be concluded that theENAC of that cell is of multimeric structure, δ1 β1 γ1. However, asthese traces are not normalized, the definitive structure may have adifferent stoichiometric ratio. The best evidence, however, suggeststhat the human salty taste receptor is composed of δ1 β1 γ1.

Once sequence confirmation is obtained, the recombinant plasmid can beused as the substrate in a process known as in vitro protein expression(IVPE). This procedure, be it either cell driven or a cell-free system,allows generation of large amounts (mM) of desired protein, in thiscase, ENaC subunits, δ, α, β, and γ. A Western blot can be used toconfirm the identity of the manufactured protein. Analysis of thereaction mixture using an antibody to the protein (a Western blot) isused to confirm that the desired protein has indeed been obtained.

The desired protein can be isolated and purified. Purification of theprotein by affinity chromatography involves chemically linking anantibody to the protein with a column matrix such as Sephadex. Passingthe IVPE reaction mixture through the column results in binding of theprotein to its antibody on the column. Elution of the column with anappropriate reagent yields the enriched protein. The protein eluate canbe quantified by measuring total nitrogen, as in the Kjeldahl procedure.This measure of total nitrogen content is then compared to the protein'sabsorption at 280 nm to calculate an absorption coefficient. From thispoint, absorption at 280 nm becomes a convenient and accurate measure ofprotein concentration.

Knowing the actual concentration of each subunit of the ENaC allows thecombination of these subunits in specific ratios, these having beenestimated by the Q-PCR of single cells. As these proteins are membraneassociated, they will require some amount of detergent to remainsoluble. While their being soluble is required for combining them inspecific ratios, detergent will destroy the lipid bilayer into whichthey need to be reconstituted to measure activity. Thus, reconstitutionof the lipid bilayer with the isolated proteins requires that anydetergent be removed. Detergent can be removed by any means suitable inthe art, such as dialysis as described herein. Reconstitution ofisolated proteins into lipid membranes has been described (Grosvenor, Wet al. (2004) BMC Neurosci. 5:2202-5), and summarized in the examplesabove. Because the subunits for human ENaC are synthesized, an advantageis gained as careful control over the composition an ratios of anyputative salt taste receptor subunits can be exerted.

The present invention is not limited to the embodiments described andexemplified above, but is capable of variation and modification withinthe scope of the appended claims.

Sequence Listing SEQ ID NO: 1 nt for human alphaatggagggga acaagctgga ggagcaggac tctagccctc cacagtccac tccagggctc atgaaggggaacaagcgtga ggagcagggg ctgggccccg aacctgcggc gccccagcag cccacggcgg aggaggaggccctgatcgag ttccaccgct cctaccgaga gctcttcgag ttcttctgca acaacaccac catccacggcgccatccgcc tggtgtgctc ccagcacaac cgcatgaaga cggccttctg ggcagtgctg tggctctgcacctttggcat gatgtactgg caattcggcc tgcttttcgg agagtacttc agctaccccg tcagcctcaacatcaacctc aactcggaca agctcgtctt ccccgcagtg accatctgca ccctcaatcc ctacaggtacccggaaatta aagaggagct ggaggagctg gaccgcatca cagagcagac gctctttgac ctgtacaaatacagctcctt caccactctc gtggccggct cccgcagccg tcgcgacctg cgggggactc tgccgcaccccttgcagcgc ctgagggtcc cgcccccgcc tcacggggcc cgtcgagccc gtagcgtggc ctccagcttgcgggacaaca acccccaggt ggactggaag gactggaaga tcggcttcca gctgtgcaac cagaacaaatcggactgctt ctaccagaca tactcatcag gggtggatgc ggtgagggag tggtaccgct tccactacatcaacatcctg tcgaggctgc cagagactct gccatccctg gaggaggaca cgctgggcaa cttcatcttcgcctgccgct tcaaccaggt ctcctgcaac caggcgaatt actctcactt ccaccacccg atgtatggaaactgctatac tttcaatgac aagaacaact ccaacctctg gatgtcttcc atgcctggaa tcaacaacggtctgtccctg atgctgcgcg cagagcagaa tgacttcatt cccctgctgt ccacagtgac tggggcccgggtaatggtgc acgggcagga tgaacctgcc tttatggatg atggtggctt taacttgcgg cctggcgtggagacctccat cagcatgagg aaggaaaccc tggacagact tgggggcgat tatggcgact gcaccaagaatggcagtgat gttcctgttg agaaccttta cccttcaaag tacacacagc aggtgtgtat tcactcctgcttccaggaga gcatgatcaa ggagtgtggc tgtgcctaca tcttctatcc gcggccccag aacgtggagtactgtgacta cagaaagcac agttcctggg ggtactgcta ctataagctc caggttgact tctcctcagaccacctgggc tgtttcacca agtgccggaa gccatgcagc gtgaccagct accagctctc tgctggttactcacgatggc cctcggtgac atcccaggaa tgggtcttcc agatgctatc gcgacagaac aattacaccgtcaacaacaa gagaaatgga gtggccaaag tcaacatctt cttcaaggag ctgaactaca aaaccaattctgagtctccc tctgtcacga tggtcaccct cctgtccaac ctgggcagcc agtggagcct gtggttcggctcctcggtgt tgtctgtggt ggagatggct gagctcgtct ttgacctgct ggtcatcatg ttcctcatgctgctccgaag gttccgaagc cgatactggt ctccaggccg agggggcagg ggtgctcagg aggtagcctccaccctggca tcctcccctc cttcccactt ctgcccccac cccatgtctc tgtccttgtc ccagccaggccctgctccct ctccagcctt gacagcccct ccccctgcct atgccaccct gggcccccgc ccatctccagggggctctgc aggggccagt tcctccacct gtcctctggg ggggccctgaSEQ ID NO: 2 pro for human alphaMEGNKLEEQD SSPPQSTPGL MKGNKREEQG LGPEPAAPQQ PTAEEEALIE FHRSYRELFE FFCNNTTIHGAIRLVCSQHN RMKTAFWAVL WLCTFGMMYW QFGLLFGEYF SYPVSLNINL NSDKLVFPAV TICTLNPYRYPEIKEELEEL DRITEQTLFD LYKYSSFTTL VAGSRSRRDL RGTLPHPLQR LRVPPPPHGA RRARSVASSLRDNNPQVDWK DWKIGFQLCN QNKSDCFYQT YSSGVDAVRE WYRFHYINIL SRLPETLPSL EEDTLGNFIFACRFNQVSCN QANYSHFHHP MYGNCYTFND KNNSNLWMSS MPGINNGLSL MLRAEQNDFI PLLSTVTGARVMVHGQDEPA FMDDGGFNLR PGVETSISMR KETLDRLGGD YGDCTKNGSD VPVENLYPSK YTQQVCIHSCFQESMIKECG CAYIFYPRPQ NVEYCDYRKH SSWGYCYYKL QVDFSSDHLG CFTKCRKPCS VTSYQLSAGYSRWPSVTSQE WVFQMLSRQN NYTVNNKRNG VAKVNIFFKE LNYKTNSESP SVTMVTLLSN LGSQWSLWFGSSVLSVVEMA ELVFDLLVIM FLMLLRRFRS RWPSPGRGGR GAQEVASTLA SSPPSHFCPH PMSLSLSQPGPAPSPALTAP PPAYATLGPR PSPGGSAGAS SSTCPLGGPSEQ ID NO: 3 nt for human betaatgcacgtga agaagtacct gctgaagggc ctgcatcggc tgcagaaggg ccccggctac acgtacaaggagctgctggt gtggtactgc gacaacacca acacccacgg ccccaagcgc atcatctgtg aggggcccaagaagaaagcc atgtggttcc tgctcaccct gctcttcgcc gccctcgtct gctggcagtg gggcatcttcatcaggacct acttgagctg ggaggtcagc gtctccctct ccgtaggctt caagaccatg gacttccccgccgtcaccat ctgcaatgct agccccttca agtattccaa aatcaagcat ttgctgaagg acctggatgagctgatggaa gctgtcctgg agagaatcct ggctcctgag ctaagccatg ccaatgccac caggaacctgaacttctcca tctggaacca cacacccctg gtccttattg atgaacggaa cccccaccac cccatggtccttgatctctt tggagacaac cacaatggct taacaagcag ctcagcatca gaaaagatct gtaatgcccacgggtgcaaa atggccatga gactatgtag cctcaacagg acccagtgta ccttccggaa cttcaccagtgctacccagg cattgacaga gtggtacatc ctgcaggcca ccaacatctt tgcacaggtg ccacagcaggagctagtaga gatgagctac cccggcgagc agatgatcct ggcctgccta ttcggagctg agccctgcaactaccggaac ttcacgtcca tcttctaccc tcactatggc aactgttaca tcttcaactg gggcatgacagagaaggcac ttccttcggc caaccctgga actgaattcg gcctgaagtt gatcctggac ataggccaggaagactacgt ccccttcctt gcgtccacgg ccggggtcag gctgatgctt cacgagcaga ggtcataccccttcatcaga gatgagggca tctacGccat gtcggggaca gagacgtcca tcggggtact cgtggacaagcttcagcgca tgggggagcc ctacagcccg tgcaccgtga atggttctga ggtccccgtc caaaacttctacagtgacta caacacgacc tactccatcc aggcctgtct tcgctcctgc ttccaagacc acatgatccgtaactgcaac tgtggccact acctgtaccc actGccccgt ggggagaaat actgcaacaa ccgggacttcccagactggg cccattgcta ctcagatcta cagatgagcg tggcgcagag agagacctgc attggcatgtgcaaggagtc ctgcaatgac acccagtaca agatgaccat ctccatggct gactggcctt ctgaggcctccgaggactgg attttccacg tcttgtctca ggagcgggac caaagcacca atatcaccct gagcaggaagggaattgtca agctcaacat ctacttccaa gaatttaact atcgcaccat tgaagaatca gcagccaataacatcgtctg gctgctctcg aatctgggtg gccagtttgg cttctggatg gggggctctg tgctgtgcctcatcgagttt ggggagatca tcatcgactt tgtgtggatc accatcatca agctggtggc cttggccaagagcctacggc agcggcgagc ccaagccagc tacgctggcc caccgcccac cgtggccgag ctggtggaggcccacaccaa ctttggcttc cagcctgaca cggccccccg cagccccaac actgggccct accccagtgagcaggccctg cccatcccag gcaccccgcc ccccaactat gactccctgc gtctgcagcc gctggacgtcatcgagtctg acagtgaggg tgatgccatc taa SEQ ID NO: 4 pro for human betaMHVKKYLLKG LHRLQKGPGY TYKELLVWYC DNTNTHGPKR IICEGPKKKA MWFLLTLLFA ALVCWQWGIFIRTYLSWEVS VSLSVGFKTM DFPAVTICNA SPFKYSKIKH LLKDLDELME AVLERILAPE LSHANATRNLNFSIWNHTPL VLIDERNPHH PMVLDLFGDN HNGLTSSSAS EKICNAHGCK MAMRLCSLNR TQCTFRNFTSATQALTEWYI LQATNIFAQV PQQELVEMSY PGEQMILACL FGAEPCNYRN FTSIFYPHYG NCYIFNWGMTEKALPSANPG TEFGLKLILD IGQEDYVPFL ASTAGVRLML HEQRSYPFIR DEGIYAMSGT ETSIGVLVDKLQRMGEPYSP CTVNGSEVPV QNFYSDYNTT YSIQACLRSC FQDHMIRNCN CGHYLYPLPR GEKYCNNRDFPDWAHCYSDL QMSVAQRETC IGMCKESCND TQYKMTISMA DWPSEASEDW IFHVLSQERD QSTNITLSRKGIVKLNIYFQ EFNYRTIEES AANNIVWLLS NLGGQFGFWM GGSVLCLIEF GEIIIDFVWI TIIKLVALAKSLRQRRAQAS YAGPPPTVAE LVEAHTNFGF QPDTAPRSPN TGPYPSEQAL PIPGTPPPNY DSLRLQPLDVIESDSEGDAI SEQ ID NO: 5 nt for human taste gammaatggcacccg gagagaagat caaagccaaa atcaagaaga atctgcccgt gacgggccct caggcgccgaccattaaaga gctgatgcgg tggtactgcc tcaacaccaa cacccatggc tgtcgccgca tcgtggtgtcccgcggccgt ctgcgccgcc tcctctggat cgggttcaca ctgactgccg tggccctcat cctctggcagtgcgccctcc tcgtcttctc cttctatact gtctcagttt ccatcaaagt ccacttccgg aagctggattttcctgcagt caccatctgc aacatcaacc cctacaagta cagcaccgtt cgccaccttc tagctgacttggaacaggag accagagagg ccctgaagtc cctgtatggc tttccagagt cccggaagcg ccgagaggcggagtcctgga actccgtctc agagggaaag cagcctagat tctcccaccg gattccgctg ctgatctttgatcaggatga gaagggcaag gccagggact tcttcacagg gaggaagcgg aaagtcggcg gtagcatcattcacaaggct tcaaatgtca tgcacatcga gtccaagcaa gtggtgggat tccaactgtg ctcaaatgacacctccgact gtgccaccta caccttcagc tcgggaatca atgccattca ggagtggtat aagctacactacatgaacat catggcacag gtgcctctgg agaagaaaat caacatgagc tattctgctg aggagctgctggtgacctgc ttctttgatg gagtgtcctg tgatgccagg aatttcacgc ttttccacca cccgatgcatgggaattgct atactttcaa caacagagaa aatgagacca ttctcagcac ctccatgggg ggcagcgaatatgggctgca agtcattttg tacataaacg aagaggaata caacccattc ctcgtgtcct ccactggagctaaggtgatc atccatcggc aggatgagta tcccttcgtc gaagatgtgg gaacagagat tgagacagcaatggtcacct ctataggaat gcacctgaca gagtccttca agctgagtga gccctacagt cagtgcacggaggacgggag tgacgtgcca atcaggaaca tctacaacgc tgcctactcg ctccagatct gccttcattcatgcttccag acaaagatgg tggagaaatg tgggtgtgcc cagtacagcc agcctctacc tcctgcagccaactactgca actaccagca gcaccccaac tggatgtatt gttactacca actgcatcga gcctttgtccaggaagagct gggctgccag tctgtgtgca aggaagcctg cagctttaaa gagtggacac taaccacaagcctggcacaa tggccatctg tggtttcgga gaagtggttg ctgcctgttc tcacttggga ccaaggccggcaagtaaaca aaaagctcaa caagacagac ttggccaaac tcttgatatt ctacaaagac ctgaaccagagatccatcat ggagagccca gccaacagta ttgagatgct tctgtccaac ttcggtggcc agctgggcctgtggatgagc tgctctgttg tctgcgtcat cgagatcatc gaggtcttct tcattgactt cttctctatcattgcccgcc gccagtggca gaaagccaag gagtggtggg cctggaaaca ggctccccca tgtccagaagctccccgtag cccacagggc caggacaatc cagccctgga tatagacgat gacctaccca ctttcaactctgctttgcac ctgcctccag ccctaggaac ccaagtgccc ggcacaccgc cccccaaata caataccttgcgcttggaga gggccttttc caaccagctc acagataccc agatgctaga tgagctctgaSEQ ID NO: 6 pro for human taste gammaMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYP FV EDVGTEIET A MVTSIGMHLT ESFKLSEP Y S QCTE DGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEAC SFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 7 nt for human kidney deltaatggctgagc accgaagcat ggacgggaga atggaagcag ccacacgggg gggctctcac ctccaggctgcagcccagac gccccccagg ccggggccac catcagcacc accaccacca cccaaggagg ggcaccaggaggggctggtg gagctgcccg cctcgttccg ggagctgctc accttcttct gcaccaatgc caccatccacggcgccatcc gcctggtctg ctcccgcggg aaccgcctca agacgacgtc ctgggggctg ctgtccctgggagccctggt cgcgctctgc tggcagctgg ggctcctctt tgagcgtcac tggcaccgcc cggtcctcatggccgtctct gtgcactcgg agcgcaagct gctcccgctg gtcaccctgt gtgacgggaa cccacgtcggccgagtccgg tcctccgcca tctggagctg ctggacgagt ttgccaggga gaacattgac tccctgtacaacgtcaacct cagcaaaggc agagccgccc tctccgccac tgtcccccgc cacgagcccc ccttccacctggaccgggag atccgtctgc agaggctgag ccactcgggc agccgggtca gagtggggtt cagactgtgcaacagcacgg gcggcgactg cttttaccga ggctacacgt caggcgtggc ggctgtccag gactggtaccacttccacta tgtggatatc ctggccctgc tgcccgcggc atgggaggac agccacggga gccaggacggccacttcgtc ctctcctgca gttacgatgg cctggactgc caggcccgac agttccggac cttccaccaccccacctacg gcagctgcta cacggtcgat ggcgtctgga cagctcagcg ccccggcatc acccacggagtcggcctggt cctcagggtt gagcagcagc ctcacctccc tctgctgtcc acgctggccg gcatcagggtcatggttcac ggccgtaacc acacgccctt cctggggcac cacagcttca gcgtccggcc agggacggaggccaccatca gcatccgaga ggacgaggtg caccggctcg ggagccccta cggccactgc accgccggcggggaaggcgt ggaggtggag ctgctacaca acacctccta caccaggcag gcctgcctgg tgtcctgcttccagcagctg atggtggaga cctgctcctg tggctactac ctccaccctc tgccggcggg ggctgagtactgcagctctg cccggcaccc tgcctgggga cactgcttct accgcctcta ccaggacctg gagacccaccggctcccctg tacctcccgc tgccccaggc cctgcaggga gtctgcattc aagctctcca ctgggacctccaggtggcct tccgccaagt cagctggatg gactctggcc acgctaggtg aacaggggct gccgcatcagagccacagac agaggagcag cctggccaaa atcaacatcg tctaccagga gctcaactac cgctcagtggaggaggcgcc cgtgtactcg gtgccgcagc tgctctccgc catgggcagc ctctacagcc tgtggtttggggcctccgtc ctctccctcc tggagctcct ggagctgctg ctcgatgctt ctgccctcac cctggtgctaggcggccgcc ggctccgcag ggcgtggttc tcctggccca gagccagccc tgcctcaggg gcgtccagcatcaagccaga ggccagtcag atgcccccgc ctgcaggcgg cacgtcagat gacccggagc ccagcgggcctcatctccca cgggtgatgc ttccaggggt tctggcggga gtctcagccg aagagagctg ggctgggccccagccccttg agactctgga cacctga Note: tac->tgc provides Tyr->Cys at 532SEQ ID NO: 8 pro for human kidney delta (with Y₅₃₂)MAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS L YSLWFGASV LSLLELLELL LDASALTLVLGGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 9 pro for humanbrain delta (with C532)MAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS L CSLWFGASV LSLLELLELL LDASALTLVLGGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 10 motif for human kidney alpha MGSQWSLWFGASEQ ID NO: 11 motif for human kidney delta MGSLYSLWFGASEQ ID NO: 12 motif for human taste bud delta MGSLCSLWFGASEQ ID NO: 13 pro for human taste gamma (Gamma A)MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPP LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS LGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYP FV EDVGTEIET A MVTSIGMHLT ESFKLSEP YS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEAC SLK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVVEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 14 pro for human taste gamma (Gamma B)MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYA VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYP FV EDVGTEIET A MVTSIGMHLT ESFKLSEP Y S QCTE DGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEAC SFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNSL TDTQMLDEL SEQ ID NO: 15 pro for human taste gamma (Gamma C)MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYP FV EDVGTEIET A MVTSIGMHLT ESFKLSEP Y S QCTE DGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEAC SFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD GLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 16 pro for human lung gamma (X87160)MAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPSV EDVGTEIETTMVTSIGMHLT ESFKLSEPSS QCTEGGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACRFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 17 pro for human delta subject DENACAMAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTIHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATIRIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVLGGRRLHRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 18 pro for human delta subject DENACDMAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVLGGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 19 pro for human delta subject DENAEMAEHRSMDGR MEAATRGGPH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPP HEPPFHLDRE IRLQSLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVLGGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDK SEQ ID NO: 20 pro for human delta subject DENACGMAEHRSMDGR MEAATRGGPH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQGLNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVLGGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 21 pro for human delta subject DENACHMAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGTIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPP HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATISIREDEV HRLGSPYGHC TAGGEGVEVQ LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYRDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVLGGRRLRRAWF SWPRASPASG ASSIKPEAGQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 22 pro for human delta subject DENACIMAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQGHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVLGGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 23 pro for human delta subject DENACJMAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPP HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATISIREDEV HRLGSPYGHC TAGGEGVEVQ PLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVLGGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 24 pro for human delta subject DENACTMAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYSVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ PCLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVLGGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 25 pro for human delta subject DENACVMAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTFHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATISIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCGSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVLGGRRLRRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 26 pro for human delta subject DENACWMAEHRSMDGR MEAATRGGSH LQAAAQTPPR PGPPSAPPPP PKEGHQEGLV ELPASFRELL TFFCTNATIHGAIRLVCSRG NRLKTTSWGL LSLGALVALC WQLGLLFERH WHRPVLMAVS VHSERKLLPL VTLCDGNPRRPSPVLRHLEL LDEFARENID SLYNVNLSKG RAALSATVPR HEPPFHLDRE IRLQRLSHSG SRVRVGFRLCNSTGGDCFYR GYTSGVAAVQ DWYHFHYVDI LALLPAAWED SHGSQDGHFV LSCSYDGLDC QARQFRTIHHPTYGSCYTVD GVWTAQRPGI THGVGLVLRV EQQPHLPLLS TLAGIRVMVH GRNHTPFLGH HSFSVRPGTEATIRIREDEV HRLGSPYGHC TAGGEGVEVE LLHNTSYTRQ ACLVSCFQQL MVETCSCGYY LHPLPAGAEYCSSARHPAWG HCFYRLYQDL ETHRLPCTSR CPRPCRESAF KLSTGTSRWP SAKSAGWTLA TLGEQGLPHQSHRQRSSLAK INIVYQELNY RSVEEAPVYS VPQLLSAMGS LCSLWFGASV LSLLELLELL LDASALTLVLGGRRLHRAWF SWPRASPASG ASSIKPEASQ MPPPAGGTSD DPEPSGPHLP RVMLPGVLAG VSAEESWAGPQPLETLDT SEQ ID NO: 27 pro for human gamma subject GENACAMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETAMVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 28 pro for human gamma subject GENACBMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVNFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETAMVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 29 pro for human gamma subject GENACDMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETAMVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 30 pro for human gamma subject GENACEMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGE ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETAMVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP STPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 31 pro for human gamma subject GENACGMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYA VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETAMVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSITARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNSL TDTQMLDEL SEQ ID NO: 32 pro for human gamma subject GENACHMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETAMVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD GLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 33 pro for human gamma subject GENACJMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPP LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS LGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETAMVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSLK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSITARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 34 pro for human gamma subject GENACTMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETAMVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 35 pro for human gamma subject GENACVMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILWQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETAMVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL SEQ ID NO: 36 pro for human gamma subject GENACWMAPGEKIKAK IKKNLPVTGP QAPTIKELMR WYCLNTNTHG CRRIVVSRGR LRRLLWIGFT LTAVALILRQCALLVFSFYT VSVSIKVHFR KLDFPAVTIC NINPYKYSTV RHLLADLEQE TREALKSLYG FPESRKRREAESWNSVSEGK QPRFSHRIPL LIFDQDEKGK ARDFFTGRKR KVGGSIIHKA SNVMHIESKQ VVGFQLCSNDTSDCATYTFS SGINAIQEWY KLHYMNIMAQ VPLEKKINMS YSAEELLVTC FFDGVSCDAR NFTLFHHPMHGNCYTFNNRE NETILSTSMG GSEYGLQVIL YINEEEYNPF LVSSTGAKVI IHRQDEYPFV EDVGTEIETAMVTSIGMHLT ESFKLSEPYS QCTEDGSDVP IRNIYNAAYS LQICLHSCFQ TKMVEKCGCA QYSQPLPPAANYCNYQQHPN WMYCYYQLHR AFVQEELGCQ SVCKEACSFK EWTLTTSLAQ WPSVVSEKWL LPVLTWDQGRQVNKKLNKTD LAKLLIFYKD LNQRSIMESP ANSIEMLLSN FGGQLGLWMS CSVVCVIEII EVFFIDFFSIIARRQWQKAK EWWAWKQAPP CPEAPRSPQG QDNPALDIDD DLPTFNSALH LPPALGTQVP GTPPPKYNTLRLERAFSNQL TDTQMLDEL

What is claimed:
 1. A method for identifying modulators of epithelialsodium ion channels, comprising: assembling at least one epithelialsodium ion channel in an artificial lipid membrane, wherein theepithelial sodium ion channel comprises at least three subunits, whereinthe subunits are independently an alpha subunit, a beta subunit, a gammasubunit, a delta subunit or an epsilon subunit; contacting the ionchannel with a test compound in the presence of sodium or lithium; anddetermining a modulation of the biological activity of the epithelialsodium ion channel in the presence of the test compound relative to thebiological activity of the epithelial sodium ion channel in the absenceof the test compound.
 2. The method of claim 1 wherein the epithelialion channel comprises at least one delta subunit, at least one betasubunit, and at least one gamma subunit.
 3. The method of claim 1,wherein the epithelial ion channel comprises at least one alpha subunit,at least one beta subunit, at least one delta, and at least one gammasubunit.
 4. The method of claim 3, further comprising at least oneepsilon subunit.
 5. The method of claim 1, further comprising contactingthe epithelial sodium ion channel with an epithelial sodium ion channelantagonist.
 6. The method of claim 1, wherein the artificial lipidmembrane is a micelle, liposome, or lipid bilayer.
 7. The method ofclaim 1, wherein at least two subunits of an epithelial sodium ionchannel are present in the lipid membrane at differing ratios relativeto each other.
 8. The method of claim 1, wherein the epithelial sodiumion channel comprises at least one biological activity of a functionalhuman salty taste receptor.
 9. A method according to claim 1, adaptedfor high throughput screening.
 10. A planar lipid bilayer comprising atleast one type of phospholipid and an epithelial sodium ion channel orspecific ratios of epithelial sodium ion channel subunits, wherein thesubunits are independently an alpha subunit, a beta subunit, a gammasubunit, a delta subunit, or an epsilon subunit.
 11. The artificiallipid membrane of claim 10, wherein the membrane is a micelle, liposome,or lipid bilayer.
 12. The planar lipid bilayer of claim 10, comprisingat least one human epithelial sodium ion channel comprising at least onealpha subunit, at least one beta subunit, at least one delta subunit,and at least one gamma subunit.
 13. The planar lipid bilayer of claim12, further comprising at least one epsilon subunit.
 14. The planarlipid bilayer of claim 10, comprising at least one epithelial sodiumchannel comprising at least one delta subunit, at least one betasubunit, and at least one gamma subunit.
 15. The planar lipid bilayer ofclaim 14, wherein the at least one epithelial sodium channel furthercomprises at least one alpha subunit.
 16. The planar lipid bilayer ofclaim 10, wherein the epithelial sodium channel comprises at least onedelta subunit.
 17. The planar lipid bilayer of claim 16, wherein theepithelial sodium channel further comprises at least one alpha subunit.18. The planar lipid bilayer of claim 10, wherein the epithelial sodiumchannel comprises at least one delta subunit, at least one beta subunit,and at least one gamma subunit wherein the ratio of the delta, beta, andgamma subunits to each other is 1:1:1.
 19. The planar lipid bilayer ofclaim 18, wherein the epithelial sodium channel further comprises atleast one alpha subunit.
 20. A method for preparing the artificial lipidmembrane of claim 10, comprising: admixing a micelle or liposomecomprising at least one phospholipid with an epithelial sodium ionchannel or specific ratios of epithelial sodium ion channel subunits,wherein the epithelial sodium ion channel or epithelial sodium ionchannel subunits are dissolved in a suitable aqueous buffer comprisingat least one surfactant; incubating said micelle or liposome with theepithelial sodium ion channel or epithelial sodium ion channel subunitfor a sufficient amount of time; and removing the at least onesurfactant.
 21. The method of claim 20, further comprisingreconstituting the proteo-liposome into a planar lipid bilayer.
 22. Amethod for identifying modulators of salty taste perception, comprising:assembling at least one epithelial sodium ion channel in an artificiallipid membrane, wherein the epithelial sodium ion channel comprises atleast one beta subunit, at least one gamma subunit, and at least onedelta subunit; contacting the ion channel with a test compound in thepresence of sodium or lithium; determining a modulation of thebiological activity of the epithelial sodium ion channel in the presenceof the test compound relative to the biological activity of theepithelial sodium ion channel in the absence of the test compound; andadministering the test compound to a subject and determining amodulation of salty taste perception in the subject relative to thelevel of salty taste perception in the subject in the absence of thetest compound.
 23. The method of claim 22 wherein the delta subunitcomprises the amino acid sequence of SEQ ID NO:12.
 24. The method ofclaim 22 further comprising the step of screening positive candidatecompounds in a cell-based assay for epithelial sodium channel activity.25. An isolated human salty taste receptor comprising at least one betapolypeptide subunit, at least one gamma polypeptide subunit and at leastone delta polypeptide subunit, wherein said delta polypeptide subunitcomprises the amino acid sequence of SEQ ID NO:12.
 26. The isolatedhuman salty taste receptor of claim 25 wherein said delta polypeptidesubunit has the amino acid sequence of SEQ ID NO:9.
 27. A kit foridentifying modulators of the human salty taste receptor comprising: atleast one phospholipid; substantially purified epithelial sodium ionchannel subunits comprising delta subunits, beta subunits, and gammasubunits; optionally comprising an epithelial sodium ion channelmodulator, sodium or lithium; and instructions for using the kit in amethod for identifying modulators of the human salty taste receptor. 28.The kit of claim 27 wherein the subunits are admixed in known ratios ina single container.
 29. The kit of claim 27 wherein at least twosubunits are present at differing ratios relative to each other.
 30. Thekit of claim 27, wherein the modulator is amiloride, phenamil, benzamil,chlorhexidine or a source of guanidinium ion or an organic polyamine.31. A compound identified by the method of claim 1, wherein the compoundactivates the epithelial sodium ion channel and induces a salty tasteperception when administered to the mouth of a subject, provided thatthe compound is not sodium or lithium.